Amide derivatives as positive allosteric modulators and methods of use thereof

ABSTRACT

The invention relates to novel amide derivatives that are positive allosteric modulators of neuronal nicotinic receptors, compositions comprising the same, processes for preparing such compounds, and methods for using such compounds and compositions.

This application is a divisional application of U.S. patent applicationSer. No.12/412,110,filed on Mar. 26, 2009, now U.S. Pat. No. 7,786,171,which in turn claims the benefit of U.S.Provisional Patent ApplicationNo. 61/042,323, filed Apr. 4, 2008, both of which are incorporatedherein by reference.

TECHNICAL FIELD

The invention relates to novel amide derivatives that are positiveallosteric modulators of neuronal nicotinic receptors, compositionscomprising the same, and methods for using such compounds andcompositions.

DESCRIPTION OF RELATED TECHNOLOGY

Nicotinic acetylcholine receptors (nAChRs), belonging to the superfamilyof ligand gated ion channels (LGIC), gate the flow of cations,controlled by acetylcholine (ACh). The nAChRs can be divided intonicotinic receptors of the muscular junction and neuronal nicotinicreceptors (NNRs). NNRs are widely distributed throughout the centralnervous system (CNS) and the peripheral nervous system (PNS). NNRs playan important role in regulating CNS function and the release of manyneurotransmitters, for example, ACh, norepinephrine, dopamine,serotonin, and GABA, among others, resulting in a wide range ofphysiological effects.

nAChRs are typically pentameric assemblies composed of protein subunitssurrounding a central ion channel. Sixteen subunits have been reportedto date, which are identified as α2-α10, β1-β4, γ, δ, and ε. Of thesesubunits, α2 through α7 and β2 through β4, are highly expressed in themammalian brain. Other functionally distinct nAChR complexes also exist,for example five α7 subunits can form a receptor as a homomericfunctional pentamer or combinations of different subunits can complextogether as in the case of α4β2 and α3β4 receptors (see for example,Paterson, D., et al., Prog. Neurobiol. 2000, 61: 75-111; Hogg, R. C., etal., Rev. Physiol., Biochem. Pharmacol., 2003, 147: 1-46; Gotti, C., etal., Prog. Neurobiol., 2004, 74: 363-396).

The homomeric α7 receptor is one of the most abundant nicotinicreceptors, along with α4β2 receptors, in the human brain, wherein it isexpressed in the hippocampus, cortex, thalamic nuclei, ventral tegmentalarea and substantia nigra (see for example, Broad, L. M., et al., Drugsof the Future, 2007, 32(2): 161-170).

The role of α7 NNRs in neuronal signaling in the CNS also has beenactively investigated (see for example, Couturier, S., et al., Neuron,1990, 5: 847-56). The α7 NNRs have been demonstrated to regulateinterneuron excitability, modulate the release of excitatory andinhibitory neurotransmitters, and lead to neuroprotective effects inexperimental in vitro models of cellular damage (see for example,Alkondon, M., et al., Prog. Brain Res., 2004, 145: 109-20).

Biophysical studies have shown that ion channels comprised of α7subunits, when expressed in heterologous expression systems, activateand desensitize rapidly, and furthermore, exhibit relatively highercalcium permeability compared to other NNR combinations (see forexample, Dajas-Bailador, F., et al, Trends Pharmacol. Sci., 2004, 25:317-24).

The NNRs, in general, are involved in various cognitive functions, suchas learning, memory, attention, and therefore in CNS disorders, i.e.,Alzheimer's disease (AD), Parkinson's disease (PD), attention deficithyperactivity disorder (ADHD), Tourette's syndrome, schizophrenia,bipolar disorder, pain, TNF-α release and inflammation, and tobaccodependence (see for example, Keller, J. J., et al., Behav. Brain Res.,2005, 162: 143-52; Gundish, D., Expert Opin. Ther. Patents, 2005, 15(9): 1221-1239; De Luca, V., et al., Acta Psychiatr. Scand., 2006, 114:211-5; Wong, H. et al Nature, 2003, 421, 384; Pavlov, V. A. et al.,Biochem. Soc. Trans., 2006, 34(6), 1037).

More particularly, α7 NNRs have been linked to conditions and disordersrelated to attention deficit disorder, ADHD, AD, mild cognitiveimpairment (MCI), senile dementia, dementia associated with Lewy bodies,dementia associated with Down's syndrome, AIDS dementia, Pick's disease,as well as cognitive deficits associated with schizophrenia (see forexample, Martin, L. F., et al., Psychopharmacology (Berl), 2004, 174:54-64; Romanelli, M. N., et al., Exp. Opin. Ther. Patents, 2007, 17(11): 1365-1377). The α7 NNRs have also been reported to slow diseaseprogression in AD (D'Andrea, M. R., et al., Curr. Pharm. Des., 2006, 12:677-84).

Accordingly, modulating the activity of α7 NNRs demonstrates promisingpotential to prevent or treat a variety of diseases indicated above,such as AD, other dementias, schizophrenia and neurodegeneration, withan underlying pathology that involves cognitive function including, forexample, aspects of learning, memory, and attention (see for example,Gotti, C., et al., Curr. Pharm. Des., 2006, 12: 407-428).

NNR ligands have been also implicated in smoking cessation, weightcontrol and as potential analgesics (see for example, Balbani, A. P. S.,et al., Exp. Opin. Ther. Patents, 2003, 13 (7): 287-297; Gurwitz, D.,Exp. Opin. Invest. Drugs, 1999, 8(6): 747-760; Vincler, M., Exp. Opin.Invest. Drugs, 2005, 14 (10): 1191-1198; Bunnelle, W. H., et al., Exp.Opin. Ther. Patents, 2003, 13 (7): 1003-1021; Decker, M. W., et al.,Exp. Opin. Invest. Drugs, 2001, 10 (10): 1819-1830; Vincler, M., et al.,Exp. Opin. Ther. Targets, 2007, 11 (7): 891-897).

Nicotine is known to provide enhanced attention and cognitiveperformance, reduced anxiety, enhanced sensory gating, and analgesia andneuroprotective effects when administered. Such effects are mediated bythe non-selective effect of nicotine at a variety of nicotinic receptorsubtypes. However, nicotine also produces adverse consequences, such ascardiovascular and gastrointestinal problems. Therefore, it is likelythat subtype-selective nicotinic ligands may have the beneficial effectsof nicotine without the undesired effects.

Examples of reported NNR ligands, such as PNU-282987 and SSR180711A, areα7 NNR agonists (see for example, Hajos, M., et al., J. Pharmacol. Exp.Ther, 2005, 312: 1213-22; Pichat, P., et al., Society for NeuroscienceAbstract, 2004, number 583.3).

Another compound, AR-R17779, has been reported to improve performance ofrats in social recognition, water mazes, or inhibitory avoidance modelsof cognitive domains (Van Kampen, M., et al., Psychopharmacology (Berl),2004, 172: 375-83). AR-R17779 also reportedly facilitates the inductionof hippocampal long-term potentiation (LTP) in a proposed cellular modelfor learning and memory in rats (Hunter, B. E., et al., Neurosci. Lett.,1994, 168: 130-4). Compound A-582941, an α7 NNR agonist, has been shownto enhance cognitive performance associated with neurodegenerativediseases such as AD and schizophrenia (Bitner, R. S., et al., J.Neuroscience, 2007, 27(39): 10578-10587).

Despite the beneficial effects of NNR ligands, it remains uncertainwhether chronic treatment with agonists affecting NNRs may providesuboptimal benefit due to sustained activation and desensitization ofthe NNR. In contrast to agonists, administering a positive allostericmodulator (PAM) can reinforce endogenous cholinergic transmissionwithout directly simulating the target receptor (see for example,Albuquerque, E. X., et al., Alzheimer Dis. Assoc. Disord., 2001, 15Suppl 1: S19-25). Nicotinic PAMs could selectively modulate the activityof ACh at α7 NNRs. Accordingly, more recently, α7 NNR-selective PAMshave emerged (see for example, Faghih, R., et al., Recent Patents on CNSDrug Discovery, 2007, 2 (2): 99-106).

Consequently, it would be beneficial to target α7 NNR function byenhancing effects of the endogenous neurotransmitter acetylcholine viaPAMs that can reinforce the endogenous cholinergic neurotransmissionwithout directly activating α7 NNRs, like agonists. Indeed, PAMs forenhancing channel activity have been proven clinically successful forGABA_(A) receptors where benzodiazepines, barbiturates, andneurosteroids behave as PAMs acting at distinct sites (see for example,Hevers, W., et al., Mol. Neurobiol., 1998, 18: 35-86).

To date, only a few NNR PAMs are known, such as 5-hydroxyindole (5-HI),ivermectin, galantamine, bovine serum albumin, and SLURP-1, a peptidederived from acetylcholinesterase (AChE). Recently, genistein, a kinaseinhibitor was reported to increase α7 responses, and PNU-120596, a ureaderivative, was reported to increase the potency and maximal efficacy ofACh as well as improve auditory gating deficits induced by amphetaminein rats. Other NNR PAMs include derivatives of quinuclidine, indole,benzopyrazole, thiazole, and benzoisothiazoles (see for example, Hurst,R. S., et al., J. Neurosci., 2005, 25: 4396-4405; Broad, L. M., et al.,Drugs of the Future, 2007, 32(2):161-170; U.S. Pat. No. 7,160,876).

Accordingly, it would be beneficial to identify and provide new NNR PAMsand compositions for treating or preventing conditions associated withα7 NNRs. It would further be particularly beneficial if such compoundscan provide improved efficacy of treatment while reducing adverseeffects associated with compounds targeting neuronal nicotinic receptorsby selectively modulating α7 NNRs.

Consequently, various embodiments of the present invention disclosenovel amide derivatives that show α7 NNR PAM activity.

SUMMARY OF THE INVENTION

An embodiment relates to compounds of formula (I):

wherein,

A is —C(O)NH— or —NHC(O)—;

R^(a), R^(b), and R^(c) are independently hydrogen, alkenyl, alkoxy,alkoxycarbonyl, alkyl, alkylcarbonyl, alkylsulfonyl, alkylthio, cyano,haloalkoxy, haloalkyl, or halogen;

R^(x) at each occurrence is independently acyloxy, alkoxy, alkyl,haloalkyl, halogen, or hydroxy;

n is 0, 1, 2, 3, or 4;

R¹ is hydrogen or halogen;

R² and R³ are independently hydrogen, alkenyl, alkoxy, alkoxycarbonyl,alkyl, alkylcarbonyl, alkylsulfonyl, alkylthio, cyano, haloalkoxy,haloalkyl, halogen or NR⁵R⁶, provided that at least one of R² or R³ isNR⁵R⁶;

R⁵ and R⁶ are independently hydrogen, alkenyl, alkoxyalkyl,alkoxycarbonyl, alkyl, alkylcarbonyl, alkylsulfonyl, arylalkyl,cycloalkyl, cycloalkylalkyl, haloalkyl, heteroarylalkyl, heterocycle, orheterocyclealkyl; or R⁵, R⁶ and the nitrogen atom to which they areattached form an optionally substituted monocyclic heteroaryl or anoptionally substituted monocyclic heterocycle;

or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.

Another embodiment relates to a method of using compounds of formula (I)or pharmaceutically acceptable salts, esters, amides or prodrugsthereof.

Yet another embodiment is directed to a method of treating conditionsand disorders that are regulated by the NNRs using compounds of formula(I) or therapeutically effective compositions of compounds of formula(I) or pharmaceutically acceptable salts, esters, amides or prodrugsthereof.

A further embodiment is directed to a method of treating a disorder orcondition that is modulated by α7 nicotinic acetylcholine receptors in apatient in need of such treatment, comprising administering atherapeutically effective amount of a compound of formula (I) orpharmaceutically acceptable salts, esters, amides or prodrugs thereof.

Another embodiment relates to a method of assessing or diagnosingconditions or disorders related to α7 NNR activity comprising allowingisotope-labeled forms of compounds of formula (I) to interact with cellsexpressing endogenous α7 NNRs or cells expressing recombinant α7 NNRsand measuring the effects of such isotope-labeled forms of compounds onsuch cells.

Various embodiments also describe the use of NNR ligands, andparticularly PAM compounds, to identify other useful target compoundsfor treating or preventing, or both, diseases or conditions associatedwith NNR function, in cell-based assays, for example in high-throughputformat, using cells or tissues that express native α7 NNR receptors forthe purpose of identifying novel α7 NNR agonists or PAMs of the α7 NNR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of a concentration response curvewhere submaximum ACh-evoked α7 current potentiation responses aremeasured in the presence of increasing concentrations of a PAM (Example17) and normalized to the effect of a reference 10 μM PAM(N′-[(2Z)-3-(2,2-difluoro-1,3-benzodioxol-5-yl)-5-methyl-1,3-thiazol-2(3H)-ylidene]-N,N-dimethylurea).

FIG. 2 is a concentration response curve obtained in the same manner fora PAM (Example 12). The response obtained by submaximum ACh in thepresence of the reference PAM at 10 μM(N′-[(2Z)-3-(2,2-difluoro-1,3-benzodioxol-5-yl)-5-methyl-1,3-thiazol-2(3H)-ylidene]-N,N-dimethylurea)is considered as 100% and the response of submaximum ACh without any PAMas 0%. Normalized PAM potentiation is plotted on the Y-axis as afunction of the concentration of the test modulator (depicted along theX-axis).

FIG. 3 is a graphical representation of nocifensive response (lickingduration) following treatment with vehicle or a PAM (Example 20)followed by exposure to formalin in mice. In Phase 2 (20-45 minutes postformalin injection) a significant reduction (68%) in licking behaviorwas observed, indicative of pain relief in this time period.

FIG. 4 is a graphical representation of the effect of vehicle or a PAM(Example 20) on sensory gating T:C (test:conditioned) ratios. A dose of0.01 μmol/kg reduced T:C ratios by 23.7±8.9%. This effect to improvesensory gating by a PAM (Example 20) is indicative that schizophrenicpatients could focus on relevant sensory stimuli, and ignore backgroundnoise.

DETAILED DESCRIPTION OF THE INVENTION Definition of Terms

For a variable that occurs more than one time in any substituent or inthe compound of the invention or any other formulae herein, itsdefinition on each occurrence is independent of its definition at everyother occurrence. Combinations of substituents are permissible only ifsuch combinations result in stable compounds. Stable compounds arecompounds which can be isolated in a useful degree of purity from areaction mixture.

As used throughout this specification and the appended claims, thefollowing terms have the following meanings.

The term “acetyl” means a —C(O)CH₃ group.

The term “acyl” means an alkyl group, as defined herein, appended to theparent molecular moiety through a carbonyl group, as defined herein.Representative examples of acyl include, but are not limited to, acetyl,1-oxopropyl, 2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl.

The term “acyloxy” means an acyl group, as defined herein, appended tothe parent molecular moiety through an oxygen atom. Representativeexamples of acyloxy include, but are not limited to, acetyloxy,propionyloxy, and isobutyryloxy.

The term “alkenyl” means a straight or branched chain hydrocarboncontaining from 2 to 10 carbons, and preferably 2, 3, 4, 5, or 6carbons, and containing at least one carbon-carbon double bond.Representative examples of alkenyl include, but are not limited to,ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl,5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

The term “alkoxy” refers to an alkyl group, as defined herein, appendedto the parent molecular moiety through an oxygen atom. Representativeexamples of alkoxy include, but are not limited to, methoxy, ethoxy,propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and thelike.

The term “alkoxyalkoxy” means an alkoxy group, as defined herein,appended to the parent molecular moiety through another alkoxy group, asdefined herein. Representative examples of alkoxyalkoxy include, but arenot limited to, tert-butoxymethoxy, 2-ethoxyethoxy, 2-methoxyethoxy, andmethoxymethoxy.

The term “alkoxyalkyl” means an alkoxy group, as defined herein,appended to the parent molecular moiety through an alkyl group, asdefined herein. Representative examples of alkoxyalkyl include, but arenot limited to, tert-butoxymethyl, 2-ethoxyethyl, 2-methoxyethyl, andmethoxymethyl.

The term “alkoxycarbonyl” means an alkoxy group, as defined herein,appended to the parent molecular moiety through a carbonyl group, asdefined herein. Representative examples of alkoxycarbonyl include, butare not limited to, methoxycarbonyl, ethoxycarbonyl, andtert-butoxycarbonyl.

The term “alkoxyimino” means an alkoxy group, as defined herein,appended to the parent molecular moiety through a —C(═NH)— group, whichalso is defined as an imino group. Representative examples ofalkoxyimino include, but are not limited to, imino(methoxy)methyl,ethoxy(imino)methyl and tert-butoxy(imino)methyl.

The term “alkoxysulfonyl” means an alkoxy group, as defined herein,appended to the parent molecular moiety through a sulfonyl group, asdefined herein. Representative examples of alkoxysulfonyl include, butare not limited to, methoxysulfonyl, ethoxysulfonyl, andpropoxysulfonyl.

The term “alkyl” means a straight or branched chain hydrocarboncontaining from 1 to 6 carbon atoms. Representative examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, and n-hexyl.

The term “alkylcarbonyl” refers to an alkyl group, as defined herein,appended to the parent molecular moiety through a carbonyl group, asdefined herein. Representative examples of alkylcarbonyl include, butare not limited to, acetyl, 1-oxopropyl, 2,2-dimethyl-1-oxopropyl,1-oxobutyl, and 1-oxopentyl.

The term “alkylcycloalkyl” means an alkyl group, as defined herein,appended to the parent molecular moiety through a cycloalkyl group, asdefined herein. Representative examples of alkylcycloalkyl include, butare not limited to, 4-ethylcyclohexyl, 3-methylcyclopentyl, and2-isopropylcyclopropyl.

The term “alkylsulfonyl” means an alkyl group, as defined herein,appended to the parent molecular moiety through a sulfonyl group, asdefined herein. Representative examples of alkylsulfonyl include, butare not limited to, methylsulfonyl and ethylsulfonyl.

The term “alkylthio” means an alkyl group, as defined herein, appendedto the parent molecular moiety through a sulfur atom. Representativeexamples of alkylthio include, but are not limited, methylthio,ethylthio, tert-butylthio, and hexylthio.

The term “amino” refers to —NR⁹⁰R⁹¹, wherein R⁹⁰ and R⁹¹ areindependently selected from hydrogen and alkyl, as defined herein.Representative examples of amino include, but are not limited to, amino,methylamino, ethylmethylamino, methylisopropylamino, dimethylamino,diisopropylamino, diethylamino, and the like.

The term “alkynyl” means a straight or branched chain hydrocarbon groupcontaining from 2 to 10 carbon atoms, and preferably 2, 3, 4, or 5carbons, and containing at least one carbon-carbon triple bond.Representative examples of alkynyl include, but are not limited to,acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and1-butynyl.

The term “amido” means an amino, alkylamino, or dialkylamino groupappended to the parent molecular moiety through a carbonyl group, asdefined herein. Representative examples of amido include, but are notlimited to, aminocarbonyl, methylaminocarbonyl, dimethylaminocarbonyl,and ethylmethylaminocarbonyl.

The term “aryl” means phenyl, a bicyclic aryl, or a tricyclic aryl. Thebicyclic aryl is naphthyl, a phenyl fused to a cycloalkyl, or a phenylfused to a cycloalkenyl. The bicyclic aryl must be attached to theparent molecular moiety through any available carbon atom containedwithin the phenyl ring. Representative examples of the bicyclic arylinclude, but are not limited to, dihydroindenyl, indenyl, naphthyl,dihydronaphthalenyl, and tetrahydronaphthalenyl. The tricyclic aryl isanthracene or phenanthrene, a bicyclic aryl fused to a cycloalkyl, abicyclic aryl fused to a cycloalkenyl, or a bicyclic aryl fused to aphenyl. The tricyclic aryl is attached to the parent molecular moietythrough any carbon atom contained within a phenyl ring. Representativeexamples of tricyclic aryl ring include, but are not limited to,azulenyl, dihydroanthracenyl, fluorenyl, and tetrahydrophenanthrenyl.

The aryl groups of this invention can be substituted with 1, 2, or 3substituents independently selected from the group consisting of alkoxy,alkyl, aryloxy, carboxy, carboxyalkyl, cycloalkyl, cycloalkoxy, halogen,haloalkoxy, haloalkyl, halothioalkoxy, hydroxyl, mercapto, thioalkoxy,thiocycloalkoxy, and thioaryloxy.

The term “arylalkyl” means an aryl group, as defined herein, appended tothe parent molecular moiety through an alkyl group, as defined herein.Representative examples of arylalkyl include, but are not limited to,benzyl, 2-phenylethyl and 3-phenylpropyl.

The term “aryloxy” means an aryl group, as defined herein, appended tothe parent molecular moiety through an oxygen atom. Representativeexamples of aryloxy include, but are not limited to, phenoxy andtolyloxy.

The term “carbonyl” means a —C(O)— group.

The term “carboxy” means a —CO₂H group.

The term “carboxyalkyl” means a carboxy group, as defined herein,appended to the parent molecular moiety through an alkyl group, asdefined herein. Representative examples of carboxyalkyl include, but arenot limited to, carboxymethyl, 2-carboxyethyl, and 3-carboxypropyl.

The term “cyano” means a —CN group, attached to the parent molecularmoiety through the carbon.

The term “cyanoalkyl” means a —CN group attached to the parent molecularmoiety through an alkyl group. Representative examples of “cyanoalkyl”include, but are not limited to, 3-cyanopropyl, and 4-cyanobutyl.

The term “cycloalkoxy” means a cycloalkyl group, as defined herein,appended to the parent molecular moiety through an oxygen atom.Representative examples of cycloalkoxy include, but are not limited to,cyclohexyloxy and cyclopropoxy.

The term “cycloalkoxyalkyl” means a cycloalkoxy group, as definedherein, appended to the parent molecular moiety through an alkyl group,wherein alkyl is as defined herein. Representative examples ofcycloalkoxylalkyl include, but are not limited to, cyclobutoxymethyl,cyclopentyloxymethyl, 2-(cyclopentyloxy)ethyl and cyclohexyloxymethyl.

The term “cycloalkyl” means a saturated cyclic hydrocarbon groupcontaining from 3 to 10 carbons. Examples of cycloalkyl includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl.

The cycloalkyls are substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9substituents independently selected from the group consisting of acyl,acyloxy, alkenyl, alkoxy, alkoxyalkoxy, alkoxyalkyl, alkoxycarbonyl,alkoxyimino, alkoxysulfonyl, alkyl, alkylcarbonyl, alkylsulfonyl,alkylthio, alkynyl, amido, carboxy, cyano, cycloalkyl, fluoroalkoxy,formyl, haloalkoxy, haloalkyl, halogen, hydroxy, hydroxyalkyl, mercapto,nitro, phenyl, oxo, —NR⁹⁸R⁹⁹, (NR⁹⁸R⁹⁹)carbonyl, —SO₂N(R⁹⁸)(R⁹⁹),—NR⁹⁸(C═O)NR⁹⁸R⁹⁹, —NR⁹⁸(C═O)Oalkyl, and —N(R⁹⁸)SO₂(R⁹⁹), wherein R⁹⁸and R⁹⁹ each are each independently selected from acyl, alkoxyalkyl,alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcycloalkyl, alkylsulfonyl,amido, aryl, cyanoalkyl, cycloalkoxyalkyl, cycloalkyl, haloalkyl,halocycloalkyl, halocycloalkylalkyl, heteroaryl, heterocycle, hydrogen,formyl, hydroxy, and hydroxyalkyl.

The term “cycloalkylalkyl” means a cycloalkyl group, as defined herein,appended to the parent molecular moiety through an alkyl group, asdefined herein. Representative examples of cycloalkylalkyl include, butare not limited to, cyclopropylmethyl, cyclopentylmethyl,cyclohexylmethyl, and cycloheptylmethyl.

The term “fluoroalkoxy” means at least one fluoro group, appended to theparent molecular moiety through an alkoxy group, as defined herein.Representative examples of fluoroalkoxy include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, pentafluoroethoxy, and2,2,2-trifluoroethoxy.

The term “formyl” means a —C(O)H group.

The term “halo” or “halogen” means —Cl, —Br, —I or —F.

The term “haloalkoxy” means at least one halogen, as defined herein,appended to the parent molecular moiety through an alkoxy group, asdefined herein. Representative examples of haloalkoxy include, but arenot limited to, 2-fluoroethoxy, trifluoromethoxy, and pentafluoroethoxy.

The term “haloalkyl” means at least one halogen, as defined herein,appended to the parent molecular moiety through an alkyl group, asdefined herein. Representative examples of haloalkyl include, but arenot limited to, difluoromethyl, chloromethyl, 2-fluoroethyl,trifluoromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, and2-chloro-3-fluoropentyl.

The term “halocycloalkyl” means at least one halogen, as defined herein,appended to the parent molecular moiety through a cycloalkyl group, asdefined herein. Representative examples of halocycloalkyl include, butare not limited to, fluorocyclohexyl, bromocyclopropyl, andtrans-2,3-dichlorocyclopentyl.

The term “halocycloalkylalkyl” means a halocycloalkyl group as definedherein, attached to the parent molecular moiety through an alkyl group.Representative examples of halocycloalkylalkyl include, but are notlimited to, (4-fluorocyclohexyl)methyl, (2,2-difluorocyclobutyl)methyland the like.

The term “halothioalkoxy” means at least one halogen, as defined herein,appended to the parent molecular moiety through a thioalkoxy group, asdefined herein. Representative examples of halothioalkoxy include, butare not limited to, 2-chloroethylsulfane and trifluoromethylsulfane.

The term “heteroaryl” means a monocyclic heteroaryl or a bicyclicheteroaryl. The monocyclic heteroaryl is a 5 or 6 membered ring thatcontains at least one heteroatom selected from the group consisting ofnitrogen, oxygen and sulfur. The 5 membered ring contains two doublebonds and the 6 membered ring contains three double bonds. The 5 or 6membered heteroaryl is connected to the parent molecular moiety throughany carbon atom or any substitutable nitrogen atom contained within theheteroaryl, provided that proper valance is maintained. Representativeexamples of monocyclic heteroaryl include, but are not limited to,furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl,pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl,tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl.The bicyclic heteroaryl consists of a monocyclic heteroaryl fused to aphenyl, or a monocyclic heteroaryl fused to a cycloalkyl, or amonocyclic heteroaryl fused to a cycloalkenyl, or a monocyclicheteroaryl fused to a monocyclic heteroaryl. The bicyclic heteroaryl isconnected to the parent molecular moiety through any carbon atom or anysubstitutable nitrogen atom contained within the bicyclic heteroaryl,provided that proper valance is maintained. Representative examples ofbicyclic heteroaryl include, but are not limited to, azaindolyl,benzimidazolyl, benzofuranyl, benzoxadiazolyl, benzoisoxazole,benzoisothiazole, benzooxazole, 1,3-benzothiazolyl, benzothiophenyl,cinnolinyl, furopyridine, indolyl, indazolyl, isobenzofuran, isoindolyl,isoquinolinyl, naphthyridinyl, oxazolopyridine,1H-pyrrolo[2,3-b]pyridinyl, quinolinyl, quinoxalinyl andthienopyridinyl.

The heteroaryl groups are optionally substituted with 1, 2, 3 or 4substituents independently selected from the group consisting ofaryloxy, alkenyl, alkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl,alkylsulfonyl, alkylthio, alkynyl, amido, aryl, aryloxy, carboxy,carboxyalkyl, cyano, cyanoalkyl, cycloalkyl, cycloalkoxy, formyl,haloalkoxy, haloalkyl, halogen, halothioalkoxy, phenyl, chlorophenyl,thioalkoxy, thiocycloalkoxy, thioaryloxy, nitro, and —NR⁷R⁸, wherein R⁷and R⁸ are independently acyl, alkoxyalkyl, alkoxycarbonyl, alkyl,alkylcarbonyl, alkylcycloalkyl, alkylsulfonyl, amido, aryl, cyanoalkyl,cycloalkoxyalkyl, cycloalkyl, haloalkyl, halocycloalkyl,halocycloalkylalkyl, heteroaryl, heterocycle, hydrogen, formyl, hydroxy,or hydroxyalkyl.

The term “heteroarylalkyl” means a heteroaryl, as defined herein,appended to the parent molecular moiety through an alkyl group, asdefined herein.

The term “heterocycle” or “heterocyclic” means a monocyclic heterocycleor a bicyclic heterocycle. The monocyclic heterocycle is a 3, 4, 5, 6 or7 membered ring containing at least one heteroatom independentlyselected from the group consisting of O, N, and S. The 3- or 4-memberedring contains 1 heteroatom selected from the group consisting of O, Nand S. The 5-membered ring contains zero or one double bond and one, twoor three heteroatoms selected from the group consisting of O, N and S.The 6- or 7-membered ring contains zero, one, or two double bondsprovided that the ring, when taken together with a substituent, does nottautomerize with a substituent to form an aromatic ring and one, two,three, or four heteroatoms selected from the group consisting of O, Nand S. The monocyclic heterocycle is connected to the parent molecularmoiety through any carbon atom or any nitrogen atom contained within themonocyclic heterocycle. Representative examples of monocyclicheterocycle include, but are not limited to, azetidinyl, azepanyl,aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl,1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl,isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl,oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl,piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl,pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl,thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl,thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone),thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclicheterocycle fused to a phenyl group, a monocyclic heterocycle fused to acycloalkyl, a monocyclic heterocycle fused to a cycloalkenyl, or amonocyclic heterocycle fused to a monocyclic heterocycle. The bicyclicheterocycle is connected to the parent molecular moiety through anycarbon atom or any nitrogen atom contained within the monocyclicheterocycle. Representative examples of bicyclic heterocycle include,but are not limited to, 1,3-benzodioxolyl, 1,3-benzodithiolyl,2,3-dihydro-1,4-benzodioxinyl, 2,3-dihydro-1-benzofuranyl,2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indolyl,hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl,tetrahydro-1H-pyrrolo[3,4-b]pyridin-6(2H,7H,7aH)-yl, and1,2,3,4-tetrahydroquinolinyl.

The heterocycles are substituted with hydrogen, or optionallysubstituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 substituentsindependently selected from acyl, acyloxy, alkenyl, alkoxy,alkoxyalkoxy, alkoxyalkyl, alkoxycarbonyl, alkoxyimino, alkoxysulfonyl,alkyl, alkylcarbonyl, alkylsulfonyl, alkylthio, alkynyl, amido, carboxy,cyano, cyanoalkyl, cycloalkyl, formyl, haloalkoxy, haloalkyl, halogen,hydroxy, hydroxyalkyl, mercapto, nitro, oxo, —NR⁹⁸R⁹⁹,(NR⁹⁸R⁹⁹)carbonyl, —SO₂N(R⁹⁸)(R⁹⁹), —NR⁹⁸(C═O)NR⁹⁸R⁹⁹, —NR⁹⁸(C═O)Oalkyl,and —N(R⁹⁸)SO₂(R⁹⁹), wherein R⁹⁸ and R⁹⁹ each are each independentlyacyl, alkoxyalkyl, alkoxycarbonyl, alkyl, alkylcarbonyl,alkylcycloalkyl, alkylsulfonyl, amido, aryl, cyanoalkyl,cycloalkoxyalkyl, cycloalkyl, haloalkyl, halocycloalkyl,halocycloalkylalkyl, heteroaryl, heterocycle, hydrogen, formyl, hydroxy,or hydroxyalkyl.

The term “heterocyclealkyl” means a heterocycle, as defined herein,appended to the parent molecular moiety through an alkyl group, asdefined herein. Representative examples of heterocyclealkyl include, butare not limited, (pyrrolidin-2-yl)methyl, 2-(morpholin-4-yl)ethyl, and(tetrahydrofuran-3-yl)methyl.

The term “hydroxy” or “hydroxyl” means an —OH group.

The term “hydroxyalkyl” means at least one hydroxy group, as definedherein, appended to the parent molecular moiety through an alkyl group,as defined herein. Representative examples of hydroxyalkyl include, butare not limited to, hydroxymethyl, 2-hydroxyethyl,2-methyl-2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypentyl, and2-ethyl-4-hydroxyheptyl.

The term “imino” means a —C(═NH)— group.

The term “mercapto” means a —SH group.

The term “nitro” means a —NO₂ group.

The term “oxo” means (═O).

The term “thioalkoxy” refers to an alkyl group, as defined herein,appended to the parent molecular moiety through a sulfur atom.Representative examples of thioalkoxy include, but are not limited to,methylthio, ethylthio, tert-butylthio, and hexylthio.

The term “thiocyloalkoxy” refers to an cycloalkyl group, as definedherein, appended to the parent molecular moiety through a sulfur atom.Representative examples of thiocylcloalkoxy include, but are not limitedto, cyclopentylsulfane and cyclohexylsulfane.

The term “thiaryloxy” means an aryl group, as defined herein, appendedto the parent molecular moiety through a sulfur atom. Representativeexamples of thioaryloxy include, but are not limited to, thiophenoxy andtolylsulfane.

The term “parenterally” refers to modes of administration, includingintravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous,intraarticular injection and infusion.

The term “Positive Allosteric Modulator (PAM)” means a compound thatenhances activity of an endogenous ligand, such as but not limited toACh, or an exogenously administered agonist.

The term “pharmaceutically acceptable salt” or “salt” refers to thosesalts which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues of humans and lower animals withoutundue toxicity, irritation, allergic response, and the like, and arecommensurate with a reasonable benefit/risk ratio and effective fortheir intended use. Pharmaceutically acceptable salts are well-known inthe art. The salts can be prepared in situ during the final isolationand purification of the compounds of the invention or separately byreacting a free base function with a suitable organic acid.Representative acid addition salts include, but are not limited toacetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups can be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates such as dimethyl, diethyl,dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl,myristyl and stearyl chlorides, bromides and iodides; arylalkyl halidessuch as benzyl and phenethyl bromides and others. Water or oil-solubleor dispersible products are thereby obtained.

Examples of acids which can be employed to form pharmaceuticallyacceptable acid addition salts include such inorganic acids ashydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acidand such organic acids as oxalic acid, maleic acid, succinic acid, andcitric acid. Basic addition salts can be prepared in situ during thefinal isolation and purification of compounds of this invention byreacting a carboxylic acid-containing moiety with a suitable base suchas the hydroxide, carbonate or bicarbonate of a pharmaceuticallyacceptable metal cation or with ammonia or an organic primary, secondaryor tertiary amine. Pharmaceutically acceptable salts include, but arenot limited to, cations based on alkali metals or alkaline earth metalssuch as lithium, sodium, potassium, calcium, magnesium, and aluminumsalts, and the like, and nontoxic quaternary ammonia and amine cationsincluding ammonium, tetramethylammonium, tetraethylammonium,methylammonium, dimethylammonium, trimethylammonium, triethylammonium,diethylammonium, ethylammonium and the like. Other representativeorganic amines useful for the formation of base addition salts includeethylenediammonium, ethanolammonium, diethanolammonium, piperidinium,and piperazinium.

The term “pharmaceutically acceptable ester” or “ester” refers to estersof compounds of the invention which hydrolyze in vivo and include thosethat break down readily in the human body to leave the parent compoundor a salt thereof. Examples of pharmaceutically acceptable, non-toxicesters of the invention include, but are not limited to, C₁-to-C₆ alkylesters and C₅-to-C₇ cycloalkyl esters. Esters of the compounds offormula (I) can be prepared according to conventional methods.Pharmaceutically acceptable esters can be appended onto hydroxy groupsby reaction of the compound that contains the hydroxy group with acidand an alkylcarboxylic acid such as acetic acid, or with acid and anarylcarboxylic acid such as benzoic acid. In the case of compoundscontaining carboxylic acid groups, the pharmaceutically acceptableesters are prepared from compounds containing the carboxylic acid groupsby reaction of the compound with base such as triethylamine and an alkylhalide, for example with methyl iodide, benzyl iodide, cyclopentyliodide or alkyl triflate. They also can be prepared by reaction of thecompound with an acid such as hydrochloric acid and an alcohol such asethanol or methanol.

The term “pharmaceutically acceptable amide” or “amide” refers tonon-toxic amides of the invention derived from ammonia, primary C₁-to-C₃alkyl amines, primary C₄-to-C₆ alkyl amines, secondary C₁-to-C₂ dialkylamines and secondary C₃-to-C₆ dialkyl amines. In the case of secondaryamines, the amine can also be in the form of a 5- or 6-memberedheterocycle containing one nitrogen atom. Amides of the compounds offormula (I) can be prepared according to conventional methods.Pharmaceutically acceptable amides can be prepared from compoundscontaining primary or secondary amine groups by reaction of the compoundthat contains the amino group with an alkyl anhydride, aryl anhydride,acyl halide, or aroyl halide. In the case of compounds containingcarboxylic acid groups, the pharmaceutically acceptable amides areprepared from compounds containing the carboxylic acid groups byreaction of the compound with base such as triethylamine, a dehydratingagent such as dicyclohexyl carbodiimide or carbonyl diimidazole, and analkyl amine, dialkylamine, for example with methylamine, diethylamine,and piperidine. They also can be prepared by reaction of the compoundwith an acid such as sulfuric acid and an alkylcarboxylic acid such asacetic acid, or with acid and an arylcarboxylic acid such as benzoicacid under dehydrating conditions such as with molecular sieves added.The composition can contain a compound of the invention in the form of apharmaceutically acceptable prodrug.

The term “pharmaceutically acceptable prodrug” or “prodrug” representsthose prodrugs of the compounds of the invention which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of humans and lower animals without undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio, and effective for their intended use. Prodrugs ofthe invention can be rapidly transformed in vivo to a parent compound offormula (I), for example, by hydrolysis in blood. A thorough discussionis provided in T. Higuchi and V. Stella, Pro-drugs as Novel DeliverySystems, V. 14 of the A.C.S. Symposium Series, and in Edward B. Roche,ed., Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press (1987).

The term “pharmaceutically acceptable carrier” or “carrier” means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are sugars such as lactose, glucose and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols such a propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;and phosphate buffer solutions; as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of one skilledin the art of formulations.

The phrase “therapeutically effective amount” means a sufficient amountof the compound to treat disorders, at a reasonable benefit/risk ratioapplicable to any medical treatment.

Although typically it may be recognized that an asterisk is used toindicate that the exact subunit composition of a receptor is uncertain,for example α3β4* indicates a receptor that contains the α3 and β4proteins in combination with other subunits, the term α7 as used hereinis intended to include receptors wherein the exact subunit compositionis both certain and uncertain. For example, as used herein α7 includeshomomeric (α7)₅ receptors and α7* receptors, which denote an NNRcontaining at least one α7 subunit.

Compounds of the Invention

An embodiment relates to compounds of formula (I):

wherein,

A is —C(O)NH— or —NHC(O)—;

R^(a), R^(b), and R^(c) are independently hydrogen, alkenyl, alkoxy,alkoxycarbonyl, alkyl, alkylcarbonyl, alkylsulfonyl, alkylthio, cyano,haloalkoxy, haloalkyl, or halogen;

R^(x) at each occurrence is independently selected from the groupconsisting of acyloxy, alkoxy, alkyl, haloalkyl, halogen, and hydroxy;

n is 0, 1, 2, 3, or 4;

R¹ is hydrogen or halogen;

R² and R³ are independently hydrogen, alkenyl, alkoxy, alkoxycarbonyl,alkyl, alkylcarbonyl, alkylsulfonyl, alkylthio, cyano, haloalkoxy,haloalkyl, halogen or NR⁵R⁶, provided that at least one of R² or R³ isNR⁵R⁶;

R⁵ and R⁶ are independently hydrogen, alkenyl, alkoxyalkyl,alkoxycarbonyl, alkyl, alkylcarbonyl, alkylsulfonyl, arylalkyl,cycloalkyl, cycloalkylalkyl, haloalkyl, heteroarylalkyl, heterocycle, orheterocyclealkyl; or R⁵, R⁶ and the nitrogen atom to which they areattached form an optionally substituted monocyclic heteroaryl or anoptionally substituted monocyclic heterocycle;

or pharmaceutically acceptable salts, esters, amides or prodrugsthereof.

Another embodiment is a compound of formula (I), wherein A is —C(O)NH—,or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein A is —NHC(O)—,or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein R^(a), R^(b)and R^(c) are independently hydrogen or halogen, or a pharmaceuticallyacceptable salt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein R^(x) isacyloxy, alkoxy, halogen, or hydroxy, and n is 1, or a pharmaceuticallyacceptable salt, ester, amide or prodrug thereof. In another embodimentof the invention, halogen is fluorine or chlorine.

Another embodiment is a compound of formula (I), wherein R¹ is hydrogen,or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein R¹ is fluorine,or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.Another embodiment is a compound of formula (I), wherein one of R² or R³is hydrogen and the other is NR⁵R⁶, wherein R⁵ and R⁶ are independentlyselected from hydrogen, alkoxycarbonyl, alkyl, alkylcarbonyl,alkylsulfonyl, arylalkyl, cycloalkyl, cycloalkylalkyl, haloalkyl,heteroarylalkyl and heterocycle, or a pharmaceutically acceptable salt,ester, amide or prodrug thereof. In another embodiment of the invention,one of R⁵ and R⁶ is alkyl and the other is hydrogen or alkyl.

Another embodiment is a compound of formula (I), wherein one of R² or R³is hydrogen and the other is NR⁵R⁶, wherein R⁵ and R⁶ and the nitrogenatom to which they are attached form a monocyclic heteroaryl, or apharmaceutically acceptable salt, ester, amide or prodrug thereof. In afurther embodiment of the invention, monocyclic heteroaryls areimidazole, pyrazole, and pyrrole.

Another embodiment is a compound of formula (I), wherein one of R² or R³is hydrogen and the other is NR⁵R⁶, wherein R⁵ and R⁶ and the nitrogenatom to which they are attached form a monocyclic heterocycle, or apharmaceutically acceptable salt, ester, amide or prodrug thereof. In anembodiment of the invention, monocyclic heterocycles are azetidine,diazepane, piperidine, and pyrrolidine.

Another embodiment is a compound of formula (I), wherein A is —C(O)NH—,R¹ is hydrogen or fluorine, and one of R² or R³ is hydrogen and theother is NR⁵R⁶, wherein R⁵ is alkyl and R⁶ is hydrogen or alkyl, or apharmaceutically acceptable salt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein A is —C(O)NH—,R¹ is hydrogen or fluorine, and one of R² or R³ is hydrogen and theother is NR⁵R⁶, wherein R⁵ is hydrogen or alkyl and R⁶ is alkylcarbonylor alkylsulfonyl, or a pharmaceutically acceptable salt, ester, amide orprodrug thereof.

Another embodiment is a compound of formula (I), wherein A is —C(O)NH—,R¹ is hydrogen or fluorine, and one of R² or R³ is hydrogen and theother is NR⁵R⁶, wherein R⁵ and R⁶ and the nitrogen atom to which theyare attached form a monocyclic heterocycle, or a pharmaceuticallyacceptable salt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein A is —C(O)NH—,R¹ is hydrogen or fluorine, and one of R² or R³ is hydrogen and theother is NR⁵R⁶, wherein R⁵ and R⁶ and the nitrogen atom to which theyare attached form a monocyclic heteroaryl, or a pharmaceuticallyacceptable salt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein A is —NHC(O)—,R¹ is hydrogen or fluorine, and one of R² or R³ is hydrogen and theother is NR⁵R⁶, wherein R⁵ and R⁶ are independently hydrogen,alkoxycarbonyl, alkyl, or haloalkyl, or a pharmaceutically acceptablesalt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein A is —NHC(O)—,R¹ is hydrogen or fluorine, and one of R² or R³ is hydrogen and theother is NR⁵R⁶, wherein R⁵ is hydrogen and R⁶ cycloalkyl or heterocycle,or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein A is —NHC(O)—,R¹ is hydrogen or fluorine, and one of R² or R³ is hydrogen and theother is NR⁵R⁶, wherein R⁵ is hydrogen and R⁶ arylalkyl,cycloalkylalkyl, or heteroarylalkyl, or a pharmaceutically acceptablesalt, ester, amide or prodrug thereof.

Another embodiment is a compound of formula (I), wherein the monocyclicheteroaryl is optionally substituted with 0, 1, 2, or 3 substituentsselected from acyloxy, alkenyl, alkoxy, alkoxycarbonyl, alkyl,alkylcarbonyl, alkylsulfonyl, alkylthio, alkynyl, amido, cyano,cyanoalkyl, cycloalkyl, haloalkoxy, haloalkyl, halogen, nitro, and—NR⁷R⁸; wherein R⁷ and R⁸ are independently acyl, alkoxyalkyl,alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcycloalkyl, alkylsulfonyl,amido, aryl, cyanoalkyl, cycloalkoxyalkyl, cycloalkyl, haloalkyl,halocycloalkyl, halocycloalkylalkyl, heteroaryl, heterocycle, hydrogen,formyl, hydroxy, or hydroxyalkyl

In a further embodiment, R⁷ and R⁸ are independently hydrogen,alkoxyalkyl, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylsulfonyl, aryl,cycloalkyl, haloalkyl, heteroaryl, or heterocycle.

Another embodiment is a compound of formula (I), wherein the monocyclicheterocycle can be optionally substituted with 0, 1, 2, or 3substituents selected from the group consisting of acyloxy, alkenyl,alkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylsulfonyl, alkylthio,alkynyl, amido, cyano, cyanoalkyl, cycloalkyl, haloalkoxy, haloalkyl,halogen, nitro, oxo, and —NR⁹⁸R⁹⁹, wherein R⁹⁸ and R⁹⁹ are independentlyhydrogen, acyl, alkoxyalkyl, alkoxycarbonyl, alkyl, alkylcarbonyl,alkylcycloalkyl, alkylsulfonyl, amido, aryl, cyanoalkyl,cycloalkoxyalkyl, cycloalkyl, haloalkyl, halocycloalkyl,halocycloalkylalkyl, heteroaryl, heterocycle, hydrogen, formyl, hydroxy,or hydroxyalkyl.

In a further embodiment, —NR⁹⁸R⁹⁹ are independently hydrogen,alkoxyalkyl, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylsulfonyl, aryl,cycloalkyl, haloalkyl, heteroaryl, or heterocycle.

Exemplary compounds of various embodiments include, but are not limitedto:

-   N-[3-(1H-pyrrol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-(3-piperidin-1-ylbenzyl)-4-[(trifluoromethyl)thio]benzamide;-   N-(3-pyrrolidin-1-ylbenzyl)-4-[(trifluoromethyl)thio]benzamide;-   N-[4-(4-methyl-1,4-diazepan-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-[4-(acetylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-[4-(diethylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-[4-(2-methyl-1H-imidazol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-{3-[(methylsulfonyl)amino]benzyl}-4-[(trifluoromethyl)thio]benzamide;-   N-[3-(methylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-[4-(2-oxopyrrolidin-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-{4-[(methylsulfonyl)amino]benzyl}-4-[(trifluoromethyl)thio]benzamide;-   N-[3-(1H-pyrazol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-[4-(3,5-dimethyl-1H-pyrazol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-[3-(dimethylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-{3-[acetyl(methyl)amino]benzyl}-4-[(trifluoromethyl)thio]benzamide;-   N-[4-(1H-pyrrol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-[4-(dimethylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   N-(4-pyrrolidin-1-ylbenzyl)-4-[(trifluoromethyl)thio]benzamide;-   N-(4-aminobenzyl)-4-[(trifluoromethyl)thio]benzamide;-   N-[4-(methylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;-   4-[(difluoromethyl)thio]-N-[4-(dimethylamino)benzyl]benzamide;-   4-[(difluoromethyl)thio]-N-[4-(methylamino)benzyl]benzamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-(4-pyrrolidin-1-ylphenyl)acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-(4-piperidin-1-ylphenyl)acetamide;-   2-(4-azetidin-1-ylphenyl)-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   2-[4-(3,3-difluoroazetidin-1-yl)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   tert-butyl    4-[2-({4-[(difluoromethyl)thio]phenyl}amino)-2-oxoethyl]phenyl    carbamate;-   2-(4-aminophenyl)-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(methylamino)phenyl]acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(dimethylamino)phenyl]acetamide;-   2-[4-(dimethylamino)phenyl]-N-{4-[(trifluoromethyl)thio]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(isobutylamino)phenyl]acetamide;-   2-{4-[(cyclohexylmethyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(2-methoxybenzyl)amino]phenyl}acetamide;-   2-{4-[(4-chlorobenzyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   2-{4-[(2-chlorobenzyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(3-methoxybenzyl)amino]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(3-methylbutyl)amino]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(quinolin-4-ylmethyl)amino]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-(4-{[(5-ethyl-2-furyl)methyl]amino}phenyl)acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(tetrahydro-2H-pyran-4-ylamino)phenyl]acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(4-phenoxybenzyl)amino]phenyl}acetamide;-   2-{4-[(cyclopropylmethyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   2-(4-{[(4-bromothien-2-yl)methyl]amino}phenyl)-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(quinolin-2-ylmethyl)amino]phenyl}acetamide;-   2-(4-{[(1-acetyl-1H-indol-3-yl)methyl]amino}phenyl)-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   2-[4-(cyclohexylamino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   2-[4-({[5-(2-chlorophenyl)-2-furyl]methyl}amino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(4-methoxybenzyl)amino]phenyl}acetamide;-   2-[4-(cyclopentylamino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   2-{4-[(3-chlorobenzyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   2-[4-(cyclobutylamino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   2-[4-(cycloheptylamino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(2-methylbutyl)amino]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-(4-{[(5-methylthien-2-yl)methyl]amino}phenyl)acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(2-naphthylmethyl)amino]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(3,3,5,5-tetramethylcyclohexyl)amino]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(thien-2-ylmethyl)amino]phenyl}acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(neopentylamino)phenyl]acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-(4-{[4-(trifluoromethyl)cyclohexyl]amino}phenyl)acetamide;-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(2,2,2-trifluoroethyl)amino]phenyl}acetamide;    and-   N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(3-phenylcyclohexyl)amino]phenyl}acetamide.

Compounds of the invention may exist as stereoisomers wherein,asymmetric or chiral centers are present. These stereoisomers are “R” or“S” depending on the configuration of substituents around the chiralelement. The terms “R” and “S” used herein are configurations as definedin IUPAC 1974 Recommendations for Section E, FundamentalStereochemistry, Pure Appl. Chem., 1976, 45: 13-30. The inventioncontemplates various stereoisomers and mixtures thereof and arespecifically included within the scope of this invention. Stereoisomersinclude enantiomers and diastereomers, and mixtures of enantiomers ordiastereomers. Individual stereoisomers of compounds of the inventionmay be prepared synthetically from commercially available startingmaterials which contain asymmetric or chiral centers or by preparationof racemic mixtures followed by resolution well-known to those ofordinary skill in the art. These methods of resolution are exemplifiedby (1) attachment of a mixture of enantiomers to a chiral auxiliary,separation of the resulting mixture of diastereomers byrecrystallization or chromatography and optional liberation of theoptically pure product from the auxiliary as described in Furniss,Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical OrganicChemistry”, 5th edition (1989), Longman Scientific & Technical, EssexCM20 2JE, England, or (2) direct separation of the mixture of opticalenantiomers on chiral chromatographic columns or (3) fractionalrecrystallization methods.

Compounds including geometric isomers of carbon-carbon double bonds andcarbon-nitrogen double bonds are included in the present invention.Substituents around a carbon-carbon or a carbon-nitrogen double bond aredesignated as being of Z or E configuration and substituents around acycloalkyl or heterocycle are designated as being of cis or transconfiguration. All geometric isomeric forms and mixtures thereof of thecompounds described herein are encompassed within the scope of thepresent invention.

Compounds of the invention can exist in radiolabeled or isotope labeledform containing one or more atoms having an atomic mass or mass numberdifferent from the atomic mass or mass number most abundantly found innature. Isotopes of atoms such as hydrogen, carbon, phosphorous, sulfur,fluorine, chlorine, and iodine include, but are not limited to, ²H, ³H,¹⁴C, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, and ¹²⁵I. Compounds that contain otherradioisotopes of these and/or other atoms are within the scope of thisinvention. In an embodiment of the invention, the isotope-labeledcompounds contain deuterium (²H), tritium (³H) or ¹⁴C radioisotopes.Isotope and radiolabeled compounds of this invention can be prepared bythe general methods well known to persons having ordinary skill in theart. Such isotope and radiolabeled compounds can be convenientlyprepared by carrying out the procedures disclosed in the above Examplesand Schemes by substituting a readily available isotope or radiolabeledreagent for a non-labeled reagent. The isotope and radiolabeledcompounds of the invention may be used as standards to determine theeffectiveness of α7 NNR ligands or modulators in the binding assays.

Amides, Esters and Prodrugs

Prodrugs are pharmacologically inactive derivatives of an active drugdesigned to ameliorate some identified, undesirable physical orbiological property. The physical properties are usually solubility (toomuch or not enough lipid or aqueous solubility) or stability related,while problematic biological properties include too rapid metabolism orpoor bioavailability which itself may be related to a physicochemicalproperty.

Prodrugs are usually prepared by: a) formation of ester, hemi esters,carbonate esters, nitrate esters, amides, hydroxamic acids, carbamates,imines, Mannich bases, and enamines of the active drug, b)functionalizing the drug with azo, glycoside, peptide, and etherfunctional groups, c) use of polymers, salts, complexes, phosphoramides,acetals, hemiacetals, and ketal forms of the drug. For example, seeAndrejus Korolkovas's, “Essentials of Medicinal Chemistry”, JohnWiley-Interscience Publications, John Wiley and Sons, New York (1988),pp. 97-118, which is incorporated in its entirety by reference herein.

Esters can be prepared from substrates of formula (I) containing eithera hydroxyl group or a carboxy group by general methods known to personsskilled in the art. The typical reactions of these compounds aresubstitutions replacing one of the heteroatoms by another atom, forexample:

Amides can be prepared from substrates of formula (I) containing eitheran amino group or a carboxy group in similar fashion. Esters can alsoreact with amines or ammonia to form amides.

Another way to make amides from compounds of formula (I) is to heatcarboxylic acids and amines together.

In Schemes 2 and 3, R and R′ are independently substrates of formula(I), alkyl or hydrogen. Various embodiments of formula (I) that aresubstrates for prodrugs, amides and esters include, but not limited to,Examples 9, 19, 20, 22, 28, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, and 62. Examples 5, 15, 27, and 46 are representative prodrugsof the invention.

Compositions of the Invention

The invention also provides pharmaceutical compositions comprising atherapeutically effective amount of a compound of formula (I) incombination with a pharmaceutically acceptable carrier. The compositionscomprise compounds of the invention formulated together with one or morenon-toxic pharmaceutically acceptable carriers. The pharmaceuticalcompositions can be formulated for oral administration in solid orliquid form, for parenteral injection or for rectal administration.

The pharmaceutical compositions of this invention can be administered tohumans and other mammals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments or drops), bucally or as an oral or nasal spray.

Pharmaceutical compositions for parenteral injection comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like, and suitable mixturesthereof), vegetable oils (such as olive oil) and injectable organicesters such as ethyl oleate, or suitable mixtures thereof. Suitablefluidity of the composition may be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions can also contain adjuvants such as preservativeagents, wetting agents, emulsifying agents, and dispersing agents.Prevention of the action of microorganisms can be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. It also can bedesirable to include isotonic agents, for example, sugars, sodiumchloride and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is oftendesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This can be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug can depend upon its rateof dissolution, which, in turn, may depend upon crystal size andcrystalline form. Alternatively, a parenterally administered drug formcan be administered by dissolving or suspending the drug in an oilvehicle.

Suspensions, in addition to the active compounds, can contain suspendingagents, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

If desired, and for more effective distribution, the compounds of theinvention can be incorporated into slow-release or targeted-deliverysystems such as polymer matrices, liposomes, and microspheres. They maybe sterilized, for example, by filtration through a bacteria-retainingfilter or by incorporation of sterilizing agents in the form of sterilesolid compositions, which may be dissolved in sterile water or someother sterile injectable medium immediately before use.

Injectable depot forms are made by forming microencapsulated matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations also are prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation also can be a sterile injectablesolution, suspension or emulsion in a nontoxic, parenterally acceptablediluent or solvent such as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, one or morecompounds of the invention is mixed with at least one inertpharmaceutically acceptable carrier such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol, and salicylic acid; b) binders such ascarboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia; c) humectants such as glycerol; d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; e) solutionretarding agents such as paraffin; f) absorption accelerators such asquaternary ammonium compounds; g) wetting agents such as cetyl alcoholand glycerol monostearate; h) absorbents such as kaolin and bentoniteclay; and i) lubricants such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using lactose or milk sugar aswell as high molecular weight polyethylene glycols.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well-known in the pharmaceutical formulatingart. They can optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, or in acertain part of the intestinal tract in a delayed manner. Examples ofmaterials useful for delaying release of the active agent can includepolymeric substances and waxes.

In an embodiment of the invention, compositions for rectal or vaginaladministration are suppositories which can be prepared by mixing thecompounds of this invention with suitable non-irritating carriers suchas cocoa butter, polyethylene glycol or a suppository wax which aresolid at ambient temperature but liquid at body temperature andtherefore melt in the rectum or vaginal cavity and release the activecompound.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. A desired compound ofthe invention is admixed under sterile conditions with apharmaceutically acceptable carrier and any needed preservatives orbuffers as may be required. Ophthalmic formulation, eardrops, eyeointments, powders and solutions are also contemplated as being withinthe scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, lactose, talc, silicic acid, aluminum hydroxide, calciumsilicates and polyamide powder, or mixtures of these substances. Sprayscan additionally contain customary propellants such aschlorofluorohydrocarbons.

Compounds and compositions of the invention also can be administered inthe form of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multi-lamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes may beused. The present compositions in liposome form may contain, in additionto the compounds of the invention, stabilizers, preservatives, and thelike. In one embodiment of the invention, lipids are the natural andsynthetic phospholipids and phosphatidylcholines (lecithins) usedseparately or together. Methods to form liposomes are known in the art.See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV,Academic Press, New York, N.Y., (1976), p 33 et seq., which isincorporated herein by reference.

Dosage forms for topical administration of a compound of this inventioninclude powders, sprays, ointments and inhalants. The active compound ismixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives, buffers or propellants. Ophthalmicformulations, eye ointments, powders and solutions are also contemplatedas being within the scope of this invention. Aqueous liquid compositionsof the invention also are particularly useful.

Yet another embodiment relates to radiolabelled or isotopically labelledpharmaceutical compositions comprising radiolabelled or isotopicallylabelled forms of compounds of formula (I).

Methods of the Invention

Compounds and compositions of the invention are useful for modulatingthe effects of NNRs, particularly by allosteric modulation. Suchcompounds can be useful for the treatment and prevention of a number ofNNR-mediated diseases or conditions.

α7 NNRs have been shown to play a significant role in enhancingcognitive function, including aspects of learning, memory and attention(Levin, E. D., J. Neurobiol., 2002, 53: 633-640). As such, α7 ligandsare suitable for the treatment of cognitive disorders including, forexample, attention deficit disorder, ADHD, AD, MCI, senile dementia,AIDS dementia, Pick's disease, dementia associated with Lewy bodies, anddementia associated with Down's syndrome, as well as cognitive deficitsassociated with schizophrenia.

In addition, α7 NNRs have been shown to be involved in theneuroprotective effects of nicotine both in vitro (Jonnala, R. B., etal., J. Neurosci. Res., 2001, 66: 565-572) and in vivo (Shimohama, S.,Brain Res., 1998, 779: 359-363). More particularly, neurodegenerationunderlies several progressive CNS disorders, including, but not limitedto, AD, Parkinson's disease, amyotrophic lateral sclerosis, Huntington'sdisease, dementia with Lewy bodies, as well as diminished CNS functionresulting from traumatic brain injury. For example, the impairedfunction of α7 NNRs by β-amyloid peptides linked to AD has beenimplicated as a key factor in development of the cognitive deficitsassociated with the disease (Liu, Q.-S., et al., PNAS, 2001, 98:4734-4739). The activation of α7 NNRs has been shown to block thisneurotoxicity (Kihara, T., J. Biol. Chem., 2001, 276: 13541-13546). Assuch, selective ligands that enhance α7 activity can counter thedeficits of Alzheimer's and other neurodegenerative diseases.

Schizophrenia is a complex disease that is characterized byabnormalities in perception, cognition, and emotions. Significantevidence implicates the involvement of α7 NNRs in this disease,including a measured deficit of these receptors in post-mortem patients(Leonard, S., Eur. J. Pharmacol., 2000, 393: 237-242). Deficits insensory processing (gating) are one of the hallmarks of schizophrenia.These deficits can be normalized by nicotinic ligands that operate atthe α7 NNR (Adler, L. E., Schizophrenia Bull., 1998, 24: 189-202;Stevens, K. E., Psychopharmacology, 1998, 136: 320-327). Thus, α7 NNRligands demonstrate potential in the treatment of schizophrenia.

A population of α7 NNRs in the spinal cord modulate serotonergictransmission that have been associated with the pain-relieving effectsof nicotinic compounds (Cordero-Erausquin, M., et al., Proc. Nat. Acad.Sci., 2001, 98: 2803-2807). The α7 NNR ligands demonstrate therapeuticpotential for the treatment of pain states, including acute pain,post-surgical pain, as well as chronic pain states includinginflammatory pain and neuropathic pain. Moreover, α7 NNRs are expressedon the surface of primary macrophages that are involved in theinflammation response, and that activation of the α7 NNR inhibitsrelease of tumor necrosis factor (TNF) and other cytokines that triggerthe inflammation response (Wang, H., Nature, 2003, 421: 384-388). TNF-αplays a pathological role in diverse inflammatory diseases includingarthritis and psoriasis and endometriosis. Therefore, selective α7 NNRligands and modulators demonstrate potential for treating conditionsinvolving inflammation and pain.

Compounds of the invention are useful for treating and preventing acondition or disorder affecting cognition, neurodegeneration, andschizophrenia. Cognitive impairment associated with schizophrenia oftenlimits the ability of patients to function normally; a symptom notadequately treated by commonly available treatments, for example,treatment with an atypical antipsychotic (Rowley, M., J. Med. Chem.,2001, 44: 477-501). Such cognitive deficit has been linked todysfunction of the nicotinic cholinergic system, in particular withdecreased activity at α7 NNR receptors (Friedman, J. I., Biol.Psychiatry, 2002, 51: 349-357). Thus, activators of α7 NNR receptors canprovide useful treatment for enhancing cognitive function inschizophrenic patients who are being treated with atypicalantipsychotics. Accordingly, the combination of an α7 NNR modulator andan atypical antipsychotic would offer improved therapeutic utility.

Examples of suitable atypical antipsychotics include, but are notlimited to, clozapine, risperidone, olanzapine, quietapine, ziprasidone,zotepine, iloperidone, and the like. Accordingly, it is contemplatedthat compounds of formula (I) also can be administered in combinationwith an atypical antipsychotic.

One of the measurable abnormalities in schizophrenic patients is the P50auditory gating deficit, an indication of impaired informationprocessing and diminished ability to “filter” unimportant or repetitivesensory information. On the basis of clinical observations that thesedeficits are normalized by nicotine, it has been suggested that the highprevalence of smoking among patients with schizophrenia (>80%) may be aform of self-medication. Pharmacological studies have shown thatnicotine's mechanism of action is via α7 NNRs. Restoration of P50 gatingdeficits in humans by α7 selective ligands, agonists and PAMs could leadto discontinuation of continuous smoking. Therefore, NNR ligands thatare selective for the α7 subtype and can be used in therapy for smokingcessation, with an improved side effect profile compared to nicotine.

An embodiment is a method of using compounds of formula (I) orpharmaceutically acceptable salts, esters, amides or prodrugs thereoffor treating or preventing conditions and disorders related to NNRmodulation or regulation in mammals. In another embodiment of theinvention, the method is useful for treating or preventing conditionsand disorders modulated by α7 NNRs modulators.

The conditions and disorders related to NNR modulation or regulationinclude, but are not limited to, attention deficit disorder, ADHD, AD,MCI, schizophrenia, senile dementia, AIDS dementia, Pick's disease,dementia associated with Lewy bodies, dementia associated with Down'ssyndrome, amyotrophic lateral sclerosis, Huntington's disease,diminished CNS function associated with traumatic brain injury, acutepain, post-surgical pain, chronic pain, inflammation, inflammatory pain,neuropathic pain, smoking cessation, depression, and various otherconditions.

Another embodiment is a method of administration of a therapeuticallyeffective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt, ester, amide or prodrug thereof to a mammal in needthereof for treating or preventing a condition or disorder selected fromattention deficit disorder, ADHD, AD, MCI, senile dementia, AIDSdementia, Pick's disease, dementia associated with Lewy bodies, dementiaassociated with Down's syndrome, amyotrophic lateral sclerosis,Huntington's disease, diminished CNS function associated with traumaticbrain injury, acute pain, post-surgical pain, chronic pain,inflammation, inflammatory pain, neuropathic pain, and depression.

Combination of exogenously applied nicotinic ligands such as α7 and α4β2NNR ligands, along with a PAM would be expected to boost beneficialeffects. Accordingly, another embodiment relates to a method of usingcompounds of formula (I) or pharmaceutically acceptable salts, esters,amides or prodrugs thereof for treating or preventing conditions anddisorders related to NNR modulation or regulation in mammals incombination with at least one of α7 NNR and α4β2 NNR ligands.

An embodiment is a method of treating a disorder or condition modulatedor regulated by α7 NNRs comprising administering the compound of formula(I) or a pharmaceutically acceptable salt, ester, amide or prodrugthereof as a stand-alone product to be combined with an α7 or α4β2 NNRligand or as a component of a fixed dose combination product with an α7or α4β2 NNR ligand.

Another embodiment is a method of administering the compositionscontaining compounds of formula (I) or pharmaceutically acceptablesalts, esters, amides or prodrugs thereof in combination with anicotinic ligand either co-dosed or in a formulation in combination witha nicotinic agonist.

Examples of nicotinic ligands include, but are not limited to,5-[(2R)-azetidin-2-ylmethoxy]-2-chloro pyridine;(3R)-1-pyridin-3-ylpyrrolidin-3-amine; 2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine;3-(5,6-dichloro-pyridin-3-yl)-(1S,5S)-3,6-diazabicyclo[3.2.0]heptane;(R,R)-1-(pyridin-3-yl)octahydro-pyrrolo[3,4-b]pyrrole;6,10-methano-6H-pyrazino[2,3-h][3]benzazepine;7,8,9,10-tetrahydro-(2S,4E)-N-methyl-5-(5-isopropoxy-3-pyridyl)-4-penten-2-amine;(2S,4E)-N-methyl-5-(5-methoxy-3-pyridyl)-4-penten-2-amine;(2S,4E)-N-methyl-5-(5-ethoxy-3-pyridyl)-4-penten-2-amine;(2S,4E)-N-methyl-3-pyrimidine-4-penten-2-amine;(5aS,8S,10aR)-5a,6,9,10-tetrahydro-7H,11H-8,10a-methanopyrido[2′,′:5,6]pyran[2,3-d]azepine;3-[1-(2,4-dimethoxy-phenyl)-meth-(E)-ylidene]-3,4,5,6-tetrahydro-[2,3]bipyridinyl;3-[1-(2-methoxy-4-hydroxyphenyl)-meth-(E)-ylidene]-3,4,5,6-tetrahydro-[2,3′]bipyridinyl;and 4-bromophenyl 1,4-diazabicyclo[3.2.2]nonane-4-carboxylate.

An embodiment relates to a method of using compositions or compounds offormula (I), or pharmaceutically acceptable salts, esters, amides orprodrugs thereof, in combination with a cholinesterase inhibitor oranother drug that increases endogenous acetylcholine release such ashistamine H3 antagonists, 5HT-6 antagonists, dopamine D1 agonists,muscarinic receptor antagonists and potassium channel blockers, leadingto potentiation of effects at the α7 nicotinic receptor subtype.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions of this invention can be varied so as to obtain an amountof the active compound(s) that is effective to achieve the desiredtherapeutic response for a particular patient, compositions and mode ofadministration. The selected dosage level will depend upon the activityof the particular compound, the route of administration, the severity ofthe condition being treated and the condition and prior medical historyof the patient being treated. However, it is within the skill of the artto start doses of the compound at levels lower than required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved.

When used in the above or other treatments, a therapeutically effectiveamount of one of the compounds can be employed in pure form or, wheresuch forms exist, in pharmaceutically acceptable salt, ester, amide orprodrug form. Alternatively, the compound can be administered as apharmaceutical composition containing the compound of the invention orpharmaceutically acceptable salt, ester, amide or prodrug in combinationwith one or more pharmaceutically acceptable carriers.

It will be understood, however, that the total daily usage of thecompounds and compositions of the invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well-known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved.

The total daily dose of the compounds of this invention administered toa human or lower animal ranges from about 0.001 mg/kg body weight toabout 1 g/kg body weight. Doses can be in the range of from about 0.001mg/kg body weight to about 100 mg/kg body weight. If desired, theeffective daily dose can be divided into multiple doses for purposes ofadministration. Consequently, single dose compositions may contain suchamounts or submultiples thereof to make up the daily dose. When used incombination, co-administered or as a fixed dose combination, the dosesmay be adjusted to achieve maximal benefit.

Another embodiment relates to a method of assessing or diagnosingconditions or disorders related to α7 NNR activity comprising allowingisotope-labeled forms of compounds of formula (I) to interact with cellsexpressing endogenous α7 NNRs or cells expressing recombinant α7 NNRs,and measuring the effects of such isotope-labeled forms of compounds onsuch cells. The radiolabeled compounds may be used as standards todetermine the effectiveness of α7 NNR ligands or modulators in thebinding assays described herein.

Various embodiments also describe the use of NNR ligands, andparticularly PAM compounds, to identify other useful target compoundsfor treating or preventing, or both, diseases or conditions associatedwith NNR function, in cell-based assays, for example in high-throughputformat, using cells or tissues that express native α7 NNRs for thepurpose of identifying novel α7 NNR agonists or PAMs of α7 NNRs, byknown protocols or as described in Determination of Biological Activitysection.

Another embodiment is a method of identifying an α7 NNR agonistcomprising allowing a compound of formula (I) to interact with cells orcell lines endogenously expressing α7 NNRs or cells expressingrecombinant α7 NNRs in a fluorescent medium and measuring changes insuch fluorescence by known protocols or as described in Determination ofBiological Activity section.

Preparation of Compounds of Formula (I)

The methods described below can entail use of various enantiomers. Thecompounds of this invention can be prepared according to the syntheticmethods described in this section, Methods of the Invention and Examplessections. Certain groups described in the Scheme are meant to illustratecertain substituents contained within the invention and are not intendedto limit the scope of the invention. Representative procedures are shownin, but are not limited to, Schemes 4-6.

As outlined in Scheme 4, compounds of formula (3) which arerepresentative of compounds of formula (I) wherein R¹, R², R³, R^(x), n,R^(a), R^(b) and R^(c) are as defined in formula (I), can be preparedaccordingly. Compounds of formula (1) when treated with compounds offormula (2) in a solvent such as N,N-dimethylformamide orN,N-dimethylacetamide in the presence of a base such asN,N-diisopropylethylamine or triethylamine and a coupling reagent suchas O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate at or near room temperature for a period of 6 to 24hours furnishes compounds of formula (3) which are representative ofcompounds of formula (I).

Alternative conditions and reagents to form compounds of formula (3)include combining an equimolar mixture of the compounds of formula (1)and compounds of formula (2) with a coupling reagent such as but notlimited to bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOPCl),1,3-dicyclohexylcarbodiimide (DCC), polymer supported1,3-dicyclohexylcarbodiimide (PS-DCC),O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU) optionally along with a coupling auxiliary such as but notlimited to 1-hydroxy-7-azabenzotriazole (HOAT) or 1-hydroxybenzotriazolehydrate (HOBT) in the presence or absence of a base such as but notlimited to N-methyl morpholine, diisopropylethylamine, and triethylaminein solvents such as, but not limited to, tetrahydrofuran,N,N-dimethylacetamide, N,N-dimethylformamide, pyridine and chloroform.Typical reactions can be carried out between 0-65° C. or may be carriedout in a microwave reactor to facilitate the coupling.

Alternatively, the carboxylic acid of formula (1) may initially beconverted to an acid chloride, typically by suspending the carboxylicacid in a solvent such as dichloromethane and then adding oxalylchloride and a catalytic amount of N,N-dimethylformamide. The solventmay be removed by evaporation, and the acid chloride redissolved inpyridine. Addition of a compound of formula (2) in the presence ofHunig's base will furnish compounds of formula (3). The reaction may beconducted at ambient or elevated temperatures over a period ranging fromseveral hours to several days.

As outlined in Scheme 5, compounds of formula (6) which arerepresentative of compounds of formula (I) wherein R¹, R², R³, R^(x), n,R^(a), R^(b) and R^(c) are as defined in formula (I), can be preparedaccordingly.

Compounds of formula (5) when treated with compounds of formula (4) in asolvent such as N,N-dimethylformamide or N,N-dimethylacetamide in thepresence of a base such as N,N-diisopropylethylamine or triethylamineand a coupling reagent such asO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate at or near room temperature for a period of 6 to 24hours furnishes compounds of formula (6) which are representative ofcompounds of formula (I).

Alternative conditions and reagents to form compounds of formula (6)include combining an equimolar mixture of the compounds of formula (4)and compounds of formula (5) with a coupling reagent such as but notlimited to bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOPCl),1,3-dicyclohexylcarbodiimide (DCC), polymer supported1,3-dicyclohexylcarbodiimide (PS-DCC),O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU) optionally along with a coupling auxiliary such as but notlimited to 1-hydroxy-7-azabenzotriazole (HOAT) or 1-hydroxybenzotriazolehydrate (HOBT) in the presence or absence of a base such as but notlimited to N-methyl morpholine, diisopropylethylamine, and triethylaminein solvents such as, but not limited to, tetrahydrofuran,N,N-dimethylacetamide, N,N-dimethylformamide, pyridine and chloroform.Typical reactions can be carried out between 0-65° C. or may be carriedout in a microwave reactor to facilitate the coupling.

Alternatively, the carboxylic acid of formula (5) may initially beconverted to an acid chloride, typically by suspending the carboxylicacid in a solvent such as dichloromethane and then adding oxalylchloride and a catalytic amount of N,N-dimethylformamide. The solventmay be removed by evaporation, and the acid chloride redissolved inpyridine. Addition of a compound of formula (5) in the presence ofHunig's base will furnish compounds of formula (6). The reaction may beconducted at ambient or elevated temperatures over a period ranging fromseveral hours to several days.

As outlined in Scheme 6, compounds of formula (9) which arerepresentative of compounds of formula (I), wherein A, R¹, R², R³,R^(x), n, R^(a), R^(b), and R^(c) are as defined in formula (I), can beprepared accordingly.

Compounds of formula (7) when treated with an aldehyde or ketone offormula (8), wherein R⁴ and R⁵ are independently hydrogen, alkyl, aryl,cycloalkyl, haloalkyl, heteroaryl or heterocycle or R⁴ and R⁵ and thecarbon atom to which they are attached form a cycloalkyl or heterocycle,in a solvent mixture such as dichloromethane and methanol (1:1) in thepresence of a reducing agent such as cyanoborohydride supported on amacroporous resin at a temperature of 20 to 60° C. in the presence ofacetic acid from 2 to 24 hours supplies compounds of formula (9) whichare representative of compounds of formula (I).

Alternative conditions and reagents to form compounds of formula (9)include combining mixtures of compounds of formula (7) and compounds offormula (8) in the presence of a reducing agent such as sodiumcyanoborohydride, sodium borohydride, and sodium triacetoxyborohydride.Hydrogen, optionally at an increased pressure, in the presence of anappropriate catalyst such as palladium on carbon or platinum on carboncan also be the reductant. Sources of hydrogen include gaseous hydrogen,formic acid, cyclodienes such as cyclohexyldiene, or a salt of formicacid such as ammonium formate. When borohydride is used as the reducingagent, it can be advantageous to conduct the reaction in the presence ofan acid such as, but not limited to, acetic acid or hydrochloric acid.Water scavenging reagents such as 4 A molecular sieves can enhance thereaction rate and completeness of the reaction. The reaction may beheated as described above to facilitate the reaction. Microwave heatingalso facilitates the reaction. Suitable solvents, for example, includeesters (e.g., ethyl acetate, isopropyl acetate, and the like), ethers(e.g., tetrahydrofuran, diethylether, 1,4-dioxane, and the like),halogenated hydrocarbons (e.g., dichloromethane, trichloromethane, andthe like), dipolar aprotic solvents (e.g., N,N-dimethylformamide,dimethyl sulfoxide and the like), and alcohols (e.g. methanol and thelike), or a mixture of solvents thereof.

In addition, nitrogen protecting groups can be used for protecting aminegroups during the synthesis of compounds of formula (I). Such methods,and some suitable nitrogen protecting groups, are described in Greeneand Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1999).For example, suitable nitrogen protecting groups include, but are notlimited to, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), benzyl(Bn), acetyl, and trifluoracetyl. More particularly, the Boc protectinggroup may be removed by treatment with an acid such as trifluoroaceticacid or hydrochloric acid. The Cbz and Bn protecting groups may beremoved by catalytic hydrogenation and acetyl and trifluoracetylprotecting groups may be removed by variety of conditions including theuse of sodium, potassium or lithium hydroxide in aqueous organic oralcoholic solvents.

The compounds and intermediates of the invention may be isolated andpurified by methods well-known to those skilled in the art of organicsynthesis. Examples of conventional methods for isolating and purifyingcompounds can include, but are not limited to, chromatography on solidsupports such as silica gel, alumina, or silica derivatized withalkylsilane groups, by recrystallization at high or low temperature withan optional pretreatment with activated carbon, thin-layerchromatography, distillation at various pressures, sublimation undervacuum, and trituration, as described for instance in “Vogel's Textbookof Practical Organic Chemistry”, 5th edition (1989), by Furniss et al.,pub. Longman Scientific & Technical, Essex CM20 2JE, England.

Some compounds of the invention have at least one basic site whereby thecompound can be treated with an acid to form a desired salt. Forexample, a compound may be reacted with an acid at or above roomtemperature to provide the desired salt, which is deposited, andcollected by filtration after cooling. Examples of acids suitable forthe reaction include, but are not limited to tartaric acid, lactic acid,succinic acid, as well as mandelic acid, atrolactic acid,methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid,naphthalenesulfonic acid, carbonic acid, fumaric acid, gluconic acid,acetic acid, propionic acid, salicylic acid, hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid, citric acid, orhydroxybutyric acid, camphorsulfonic acid, malic acid, phenylaceticacid, aspartic acid, glutamic acid, and the like.

The invention contemplates pharmaceutically active compounds eitherchemically synthesized or formed by in vivo biotransformation tocompounds of formula (I). The compounds, compositions, and methods ofthe invention will be better understood by reference to the followingexamples and reference examples, which are intended as an illustrationof and not a limitation upon the scope of the invention.

EXAMPLES Abbreviations

ACh for acetylcholine, DMSO for dimethyl sulfoxide, ERK forextracellular signal-regulated kinase, HPLC for high-pressure liquidchromatography, HEPES for 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid, PAM for positive allosteric modulator, PBS for phosphate bufferedsaline, SDS for sodium dodecyl sulfate, and Tween forpolyoxoethylenesorbitan monolaurate.

General Procedure for Amide Formation (Method A):

In a 96 deep well plate, 4-(trifluoromethylthio)benzoic acid (23 mg,0.10 mmol) was added dissolved in N,N-dimethylacetamide (0.4 mL),followed by O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (48 mg, 0.12 mmol, 1.2 equivalents) dissolvedin N,N-dimethylacetamide (0.3 mL), triethylamine (26 mg, 0.25 mmol, 2.4equivalents) dissolved in N,N-dimethylacetamide (0.3 mL), and finallythe amine (0.7 mL of a 0.2 M solution in N,N-dimethylacetamide, 1.4equivalents). This was shaken at room temperature overnight. Thereaction solution was concentrated to dryness in vacuo, dissolved in 1:1dimethyl sulfoxide/methanol and purified by reverse phase HPLC on aPhenomenex® Luna® Combi-HTS C8(2) 5 μm 100 Å (2.1 mm×30 mm) using agradient of 10-100% acetonitrile (A) and 0.1% trifluoroacetic acid (B)in water at a flow rate of 2.0 mL/minute (0-0.1 minutes 10% A, 0.1-2.6minutes 10-100% A, 2.6-2.9 minutes 100% A, 2.9-3.0 minutes 100-10% A.)to give the product.

General Procedure for Amide Formation (Method B):

A suspension of an amine (0.5 mmol) and a benzoic acid (0.5 mmol) inanhydrous N,N-dimethylformamide (2 mL) was treated withN,N-diisopropylethylamine (iPr₂NEt; 245 μL, 1.5 mmol, 3.0 equivalents;Acros) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluoro-phosphate (HATU; 285 mg, 0.75 mmol, 1.5 equivalents;Aldrich). The mixture was stirred overnight at room temperature, anddiluted with dichloromethane (20 mL). The solution was washed withdilute aqueous ammonium chloride (2×7 mL), dilute aqueous sodiumbicarbonate (2×7 mL), and brine (2×7 mL). The organic layer was driedover sodium sulfate, filtered, and concentrated in vacuo. The resultingmaterial was purified by either flash chromatography [Analogixpre-packed silica gel cartridges, 5-50% gradient of ethylacetate/hexanes], or by preparative HPLC [Waters® Xterra® RP₁₈ 30×100 mmcolumn, flow rate 40 mL/minute, 5-95% gradient of acetonitrile in buffer(0.1 M aqueous ammonium bicarbonate, adjusted to pH 10 with sodiumhydroxide)] to afford the desired amide product as its free base.Alternatively, the compound was purified on a Waters® Symmetry® C₈30×100 mm column (flow rate 40 mL/minute, 5-95% gradient of acetonitrilein 0.1% aqueous trifluoroacetic acid) to afford the amide product afterevaporation of solvent.

General Procedure for Amide Formation (Method C):

A solution of a phenylacetic acid (0.5 mmol, 1 equivalent) andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU; 285 mg, 0.75 mmol, 1.5 equivalent) inanhydrous N,N-dimethylformamide (2 mL) was treated withN,N-diisopropylethylamine (iPr₂NEt; 245 μL, 1.5 mmol, 3.0 equivalent)and 4-(difluoromethylthio)aniline (0.5 mmol, 1 equivalent). The mixturewas stirred overnight at room temperature, and diluted with ethylacetate (20 mL). The solution was washed with dilute aqueous ammoniumchloride (2×7 mL), dilute aqueous sodium bicarbonate (2×7 mL), and brine(2×7 mL). The organic layer was dried over sodium sulfate, filtered, andconcentrated in vacuo. The resulting material was purified by eitherflash chromatography [Analogix pre-packed silica gel cartridges, 5-50%gradient of ethyl acetate/hexanes], or by preparative HPLC [Waters®Xterra® RP₁₈ 30×100 mm column, flow rate 40 mL/minute, 5-95% gradient ofacetonitrile in buffer (0.1 M aqueous ammonium bicarbonate, adjusted topH 10 with sodium hydroxide)] to afford the desired amide product as itsfree base.

Alternatively, the compound was purified on a Waters® Symmetry® C₈30×100 mm column (flow rate 40 mL/minute, 5-95% gradient of acetonitrilein 0.1% aqueous trifluoroacetic acid) to afford the amide product afterevaporation of solvent.

General Procedure for Reductive Amination (Method D):

To a solution of2-(4-aminophenyl)-N-{4-[(difluoromethyl)thio]phenyl}acetamidetrifluoroacetate (Example 28) (50 mg, 0.12 mmol in 1.0 mLmethanol/dichloromethane; 1:1) was added a solution of the aldehyde orketone (0.14 mmol in 1.0 mL methanol/dichloromethane; 1:1). To thismixture, acetic acid (34 μL, 0.59 mmol) and macroporous resin supportedcyanoborohydride (2.24 mmol/g, 158 mg, 0.36 mmol) were added and themixture was shaken at 55° C. overnight. After cooling, the resin wasremoved by filtration, washed with methanol (2×2 mL), and the combinedfiltrate was concentrated under reduced pressure. The crude product waspurified by reverse-phase preparative HPLC [Phenomenex® Luna® C8(2) 5 μm100 Å AXIA™ column (30 mm×75 mm), (flow rate 50 mL/min, 10-100% gradientof acetonitrile in 0.1% aqueous trifluoroacetic acid)] to furnish theproduct after evaporation of the solvents.

Example 1N-[3-(1H-pyrrol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from (3-(1H-pyrrol-1-yl)phenyl)methanamineaccording to Method A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 4.54-4.58 (m,2H), 6.28 (t, J=2.1 Hz, 2H), 7.22 (d, J=6.4 Hz, 1H), 7.32 (t, J=2.1 Hz,2H), 7.38-7.47 (m, 2H), 7.51 (s, 1H), 7.81-7.87 (m, J=8.2 Hz, 2H),7.96-8.03 (m, J=8.2 Hz, 2H), 9.32 (t, J=6.0 Hz, 1H); MS (ESI+) m/z 377.0(M+H)⁺.

Example 2 N-(3-piperidin-1-ylbenzyl)-4-[(trifluoromethyl)thio]benzamide

The product was prepared from (3-(piperidin-1-yl)phenyl)methanamineaccording to Method A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.63-1.71 (m,2H), 1.85-1.93 (m, 4H), 3.46-3.55 (m, 4H), 4.56 (s, 2H), 7.44 (t, J=3.7Hz, 1H), 7.55 (d, J=4.3 Hz, 2H), 7.59 (s, 1H), 7.83-7.88 (m, J=8.2 Hz,2H), 7.99-8.03 (m, J=8.2 Hz, 2H), 9.36 (t, J=5.8 Hz, 1H); MS (ESI+) m/z395.1 (M+H)⁺.

Example 3 N-(3-pyrrolidin-1-ylbenzyl)-4-[(trifluoromethyl)thio]benzamide

The product was prepared from (3-(pyrrolidin-1-yl)phenyl)methanamineaccording to Method A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.90-1.99 (m,4H), 3.18-3.26 (m, 4H), 4.43 (s, 2H), 6.51 (dd, J=7.9, 1.8 Hz, 1H), 6.60(s, 1H), 6.63 (d, J=7.6 Hz, 1H), 7.15 (t, J=7.8 Hz, 1H), 7.82-7.86 (m,J=8.2 Hz, 2H), 7.93-8.04 (m, J=8.5 Hz, 2H); MS (ESI+) m/z 381.0 (M+H)⁺.

Example 4N-[4-(4-methyl-1,4-diazepan-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from(4-(4-methyl-1,4-diazepan-1-yl)phenyl)methanamine according to Method A:¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.03-2.23 (m, 2H), 2.84 (s, 3H), 3.12(ddd, J=12.5, 11.2, 1.2 Hz, 1H), 3.18 (ddd, J=12.4, 10.7, 1.1 Hz, 1H),3.35-3.48 (m, 2H), 3.52 (dd, J=13.0, 3.8 Hz, 1H), 3.60 (dd, J=15.6, 9.2Hz, 1H), 3.72-3.79 (m, 2H), 4.37 (s, 2H), 6.70-6.76 (m, J=8.8 Hz, 2H),7.16-7.22 (m, J=8.8 Hz, 2H), 7.79-7.85 (m, J=8.5 Hz, 2H), 7.93-7.98 (m,J=8.2 Hz, 2H); MS (ESI+) m/z 424.0 (M+H)⁺.

Example 5 N-[4-(acetylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from N-(4-(aminomethyl)phenyl)acetamideaccording to Method A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.03 (s, 3H),4.44 (d, J=5.2 Hz, 2H), 7.21-7.29 (m, J=8.8 Hz, 2H), 7.47-7.53 (m, J=8.5Hz, 2H), 7.80-7.85 (m, J=8.2 Hz, 2H), 7.92-8.01 (m, J=8.5 Hz, 2H), 9.23(t, J=6.0 Hz, 1H); MS (ESI+) m/z 369.0 (M+H)⁺.

Example 6 N-[4-(diethylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from 4-(aminomethyl)-N,N-diethylanilineaccording to Method A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.01 (t, J=7.2Hz, 6H), 3.58 (q, J=7.2 Hz, 4H), 4.56 (s, 2H), 7.50-7.57 (m, 4H),7.83-7.88 (m, J=8.2 Hz, 2H), 7.97-8.03 (m, J=8.5 Hz, 2H), 9.36 (t, J=6.0Hz, 1H); MS (ESI+) m/z 383.1 (M+H)⁺.

Example 7N-[4-(2-methyl-1H-imidazol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from(4-(2-methyl-1H-imidazol-1-yl)phenyl)methanamine according to Method A:¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.52 (s, 3H), 4.60 (s, 2H), 7.56-7.62(m, 4H), 7.71 (d, J=2.1 Hz, 1H), 7.80 (d, J=2.1 Hz, 1H), 7.84-7.89 (m,J=8.2 Hz, 2H), 7.98-8.05 (m, J=8.5 Hz, 2H), 9.43 (t, J=6.0 Hz, 1H); MS(ESI+) m/z 392.1 (M+H)⁺.

Example 8N-{3-[(methylsulfonyl)amino]benzyl}-4-[(trifluoromethyl)thio]benzamide

The product was prepared fromN-(3-(aminomethyl)phenyl)methanesulfonamide according to Method A: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 2.97 (s, 3H), 4.48 (s, 2H), 7.10 (t, J=8.5Hz, 2H), 7.18 (s, 1H), 7.32 (t, J=7.8 Hz, 1H), 7.81-7.87 (m, J=8.2 Hz,2H), 7.96-8.01 (m, J=8.5 Hz, 2H), 9.28 (t, J=6.3 Hz, 1H), 9.73 (s, 1H);MS (ESI+) m/z 405.0 (M+H)⁺.

Example 9 N-[3-(methylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from 3-(aminomethyl)-N-methylaniline accordingto Method A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.87 (s, 3H), 4.51 (s, 2H),7.13 (d, J=7.9 Hz, 1H), 7.19 (s, 1H), 7.21 (d, J=7.9 Hz, 1H), 7.41 (t,J=7.8 Hz, 1H), 7.85 (m, J=8.2 Hz, 2H), 8.00 (m, J=8.5 Hz, 2H), 9.32 (t,J=6.3 Hz, 1H); MS (ESI+) m/z 341.0 (M+H)⁺.

Example 10N-[4-(2-oxopyrrolidin-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from 1-(4-(aminomethyl)phenyl)pyrrolidin-2-oneaccording to Method A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.06 (dt, J=15.2,7.5 Hz, 2H), 2.49 (t, J=8.1 Hz, 2H), 3.82 (t, J=7.0 Hz, 2H), 4.46 (s,2H), 7.30-7.36 (m, J=8.5 Hz, 2H), 7.55-7.61 (m, J=8.8 Hz, 2H), 7.81-7.85(m, J=8.5 Hz, 2H), 7.94-8.02 (m, J=8.5 Hz, 2H), 9.26 (t, J=6.1 Hz, 1H);MS (ESI+) m/z 395.0 (M+H)⁺.

Example 11N-{4-[(methylsulfonyl)amino]benzyl}-4-[(trifluoromethyl)thio]benzamide

The product was prepared fromN-(4-(aminomethyl)phenyl)methanesulfonamide according to Method A: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 2.95 (s, 3H), 4.46 (s, 2H), 7.14-7.21 (m,J=8.5 Hz, 2H), 7.28-7.35 (m, J=8.5 Hz, 2H), 7.81-7.85 (m, J=8.2 Hz, 2H),7.96-8.01 (m, J=8.5 Hz, 2H), 9.25 (t, J=6.0 Hz, 1H), 9.67 (s, 1H); MS(ESI+) m/z 405.0 (M+H)⁺.

Example 12N-[3-(1H-pyrazol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from (3-(1H-pyrazol-1-yl)phenyl)methanamineaccording to Method A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 4.58 (s, 2H),6.56 (dd, J=2.4, 1.8 Hz, 1H), 7.29 (d, J=7.9 Hz, 1H), 7.47 (t, J=7.8 Hz,1H), 7.70 (dd, J=8.1, 1.4 Hz, 1H), 7.75 (d, J=1.5 Hz, 1H), 7.81 (s, 1H),7.83-7.87 (m, J=8.2 Hz, 2H), 7.98-8.03 (m, J=8.5 Hz, 2H), 8.43 (d, J=2.4Hz, 1H), 9.36 (t, J=5.8 Hz, 1H); MS (ESI+) m/z 378.0 (M+H)⁺.

Example 13N-[4-(3,5-dimethyl-1H-pyrazol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from(4-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)methanamine according to MethodA: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.18 (s, 3H), 2.27 (s, 3H), 4.56 (s,2H), 6.10 (s, 1H), 7.41-7.48 (m, 4H), 7.83-7.87 (m, J=8.2 Hz, 2H),7.98-8.03 (m, J=8.2 Hz, 2H), 9.35 (t, J=6.0 Hz, 1H); MS (ESI+) m/z 406.0(M+H)⁺.

Example 14N-[3-(dimethylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from 3-(dimethylamino)benzylamine according toMethod A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.09 (s, 6H), 4.52 (s, 2H),7.19 (d, J=7.6 Hz, 1H), 7.25 (d, J=8.2 Hz, 1H), 7.29 (s, 1H), 7.43 (t,J=7.8 Hz, 1H), 7.81-7.89 (m, J=8.2 Hz, 2H), 7.94-8.05 (m, J=8.5 Hz, 2H),9.32 (t, J=5.8 Hz, 1H); MS (ESI+) m/z 355.0 (M+H)⁺.

Example 15N-{3-[acetyl(methyl)amino]benzyl}-4-[(trifluoromethyl)thio]benzamide

The product was prepared fromN-(3-(aminomethyl)phenyl)-N-methylacetamide according to method A: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 1.77 (s, 3H), 3.15 (s, 3H), 4.53 (s, 2H),7.22 (d, J=7.0 Hz, 1H), 7.27 (s, 1H), 7.32 (d, J=6.1 Hz, 1H), 7.43 (t,J=7.2 Hz, 1H), 7.81-7.86 (m, J=8.2 Hz, 2H), 7.97-8.02 (m, J=8.2 Hz, 2H),9.31 (t, J=6.1 Hz, 1H); MS (ESI+) m/z 383.1 (M+H)⁺.

Example 16N-[4-(1H-pyrrol-1-yl)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from (4-(1H-pyrrol-1-yl)phenyl)methanamineaccording to Method A: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 4.51 (s, 2H),6.27 (t, J=2.1 Hz, 2H), 7.31 (t, J=2.1 Hz, 2H), 7.40-7.44 (m, J=8.5 Hz,2H), 7.50-7.54 (m, J=8.5 Hz, 2H), 7.82-7.86 (m, J=8.2 Hz, 2H), 7.97-8.03(m, J=8.5 Hz, 2H), 9.31 (t, J=6.1 Hz, 1H); MS (ESI+) m/z 377.0 (M+H)⁺.

Example 17N-[4-(dimethylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from 4-trifluoromethylthiobenzoic acid (133 mg)and 4-(dimethylamino)benzylamine dihydrochloride (134 mg) according toMethod B: ¹H NMR (300 MHz, DMSO-d₆) δ ppm 2.85 (s, 6H), 4.36 (d, J=5.8Hz, 2H), 6.57-6.75 (m, J=8.8 Hz, 2H), 7.08-7.23 (m, J=8.8 Hz, 2H),7.76-7.86 (m, J=8.5 Hz, 2H), 7.92-8.06 (m, J=8.5 Hz, 2H), 9.08 (t, J=5.8Hz, 1H); MS (DCI/NH₃) m/z 355.1 (M+H)⁺; Anal. C₁₇H₁₇F₃N₂OS: C, H, N.

Example 18N-(4-pyrrolidin-1-ylbenzyl)-4-[(trifluoromethyl)thio]benzamide

The product was prepared from 4-trifluoromethylthiobenzoic acid (133 mg)and (4-(pyrrolidin-1-yl)phenyl)methanamine (127 mg) according to MethodB: ¹H NMR (300 MHz, DMSO-d₆) δ ppm 1.84-1.99 (m, 4H), 3.12-3.23 (m, 4H),4.35 (d, J=6.1 Hz, 2H), 6.49 (m, J=8.8 Hz, 2H), 7.13 (m, J=8.8 Hz, 2H),7.81 (m, J=8.1 Hz, 2H), 7.93-8.00 (m, 2H), 9.06 (t, J=5.9 Hz, 1H); MS(DCI/NH₃) m/z 381.1 (M+H)⁺.

Example 19 N-(4-aminobenzyl)-4-[(trifluoromethyl)thio]benzamide

The product was prepared from 4-trifluoromethylthiobenzoic acid (444 mg)and 4-(aminomethyl)aniline (342 mg) according to Method B: ¹H NMR (300MHz, DMSO-d₆) δ ppm 4.30 (d, J=6.1 Hz, 2H), 4.95 (s, 2H), 6.47-6.54 (m,J=8.5 Hz, 2H), 6.93-7.02 (m, J=8.1 Hz, 2H), 7.77-7.84 (m, J=8.1 Hz, 2H),7.93-8.02 (m, J=8.5 Hz, 2H), 9.02 (t, J=5.9 Hz, 1H); MS (ESI+) m/z 326.9(M+H)⁺; Anal. C₁₅H₁₃F₃N₂OS: C, H, N.

Example 20 N-[4-(methylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide

The product was prepared from 4-trifluoromethylthiobenzoic acid (61 mg)and 4-(aminomethyl)-N-methylaniline (128 mg) according to Method B: ¹HNMR (300 MHz, CDCl₃) δ ppm 2.84 (s, 3H) 3.76 (s, 1H) 4.52 (d, J=5.2 Hz,2H) 6.24 (s, 1H) 6.56-6.63 (m, 2H) 7.14-7.21 (m, 2H) 7.67-7.72 (m, 2H)7.76-7.83 (m, 2H); MS (DCI/NH₃) m/z 341.1 (M+H)⁺.

Example 21 4-[(difluoromethyl)thio]-N-[4-(dimethylamino)benzyl]benzamide

A sealed tube was charged with sodium 2-chloro-2,2-difluoroacetate(0.525 g, 3.44 mmol) and sodium bicarbonate (0.284 g, 3.38 mmol) inN,N-dimethylformamide (14 mL). The vessel was purged with nitrogen, and4-mercaptobenzoic acid (0.35 g, 2.270 mmol) was added. The mixture waswarmed to 80° C. for 3 hours. After 3 hours, the reaction mixture wascooled and filtered to remove a precipitate. The N,N-dimethylformamidewas diluted with 200 mL of diethyl ether and 75 mL of dichloromethaneand acidified to pH 4.8 with 0.5 mL of acetic acid. The resulting cloudysolution was extracted with 3×150 mL of aqueous sodium chloride and2×150 mL of brine. The organic layer was concentrated onto silica forloading onto a column. The product was collected after filtrationthrough silica (40 g column, 1:1 ethyl acetate/hexane eluent). The crudeproduct, 4-(difluoromethylthio)benzoic acid, was used as such in thenext step. ¹H NMR (300 MHz, CD₃OD) δ ppm 7.21 (t, J=56.3 Hz, 1H)7.62-7.70 (AA′BB′,2H) 8.00-8.08 (AA′BB′,2H); MS (ESI−) m/z 202.8 (M−H)⁻.

The product was prepared from 4-difluoromethylthiobenzoic acid (61 mg)and (4-(aminomethyl)-N,N-dimethylaniline (45 mg) according to Method B:¹H NMR (300 MHz, DMSO-d₆) δ ppm 2.85 (s, 6H), 4.36 (d, J=5.9 Hz, 2H),6.68 (d, J=8.7 Hz, 2H), 7.14 (d, J=8.7 Hz, 2H), 7.57 (t, J=56 Hz, 1H),7.65 (d, J=8.3 Hz, 2H), 7.92 (d, J=8.3 Hz, 2H), 9.00 (t, J=5.8 Hz, 1H);MS (DCI/NH₃) m/z 337.1 (M+H)⁺; Anal. C₁₇H₁₈F₂N₂OS: C, H, N.

Example 22 4-[(difluoromethyl)thio]-N-[4-(methylamino)benzyl]benzamide

The titled compound was prepared from 4-difluoromethylthiobenzoic acid(prepared as in Example 22) (150 mg) and 4-(aminomethyl)-N-methylaniline(120 mg) according to Method B: ¹H NMR (300 MHz, CDCl₃) δ ppm 2.84 (s,3H) 4.52 (d, J=5.09 Hz, 2H) 6.60 (m, J=8.48 Hz, 2H) 6.85 (t, J=56.45 Hz,1H) 7.18 (m, J=8.48 Hz, 2H) 7.61 (m, J=8.14 Hz, 2H) 7.71-7.83 (m, 2H);MS (DCI/NH₃) m/z 323 (M+H)⁺, 340 (M+NH₄)⁺.

Example 23N-{4-[(difluoromethyl)thio]phenyl}-2-(4-pyrrolidin-1-ylphenyl)acetamide

The product was prepared from 2-(4-(pyrrolidin-1-yl)phenyl)acetic acid(133 mg) according to Method C: ¹H NMR (300 MHz, CD₃OD) δ ppm 1.97-2.03(m, 4H), 3.19-3.27 (m, 4H), 3.55 (s, 2H), 6.51-6.58 (m, 2H), 6.99 (t,J=56.7 Hz, 1H), 7.10-7.17 (m, 2H), 7.47-7.54 (m, 2H), 7.60-7.66 (m, 2H);MS (DCI/NH₃) m/z 363.0 (M+H)⁺.

Example 24N-{4-[(difluoromethyl)thio]phenyl}-2-(4-piperidin-1-ylphenyl)acetamide

The product was prepared from 2-(4-(piperidin-1-yl)phenyl)acetic acid(65 mg) according to Method C: ¹H NMR (300 MHz, CDCl₃) δ ppm 1.56-1.65(m, 2H), 1.67-1.79 (m, 4H), 3.15-3.23 (m, 4H), 3.66 (s, 2H), 6.74 (t,J=57.1 Hz, 1H), 6.94-6.98 (m, 2H), 7.13 (s, 1H), 7.15-7.20 (m, 2H),7.40-7.52 (m, 4H); MS (DCI/NH₃) m/z 377.1 (M+H)⁺.

Example 252-(4-azetidin-1-ylphenyl)-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared from 2-(4-(azetidin-1-yl)phenyl)acetic acid(293 mg) according to Method C: ¹H NMR (300 MHz, CDCl₃) δ ppm 2.41 (dt,J=14.6, 7.3 Hz, 2H), 3.65 (s, 2H), 3.93 (t, J=7.1 Hz, 4H), 6.47-6.54 (m,2H), 6.74 (t, J=57.1 Hz, 1H), 7.12-7.19 (m, 2H), 7.41-7.51 (m, 4H); MS(DCI/NH₃) m/z 349.1 (M+H)⁺.

Example 262-[4-(3,3-difluoroazetidin-1-yl)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared from2-[4-(3,3-difluoroazetidin-1-yl)phenyl]acetic acid (133 mg) according toMethod C: ¹H NMR (300 MHz, CDCl₃) δ ppm 3.67 (s, 2H), 4.24 (t, J=11.7Hz, 4H), 6.51-6.55 (m, 2H), 6.74 (t, J=57.0 Hz, 1H), 7.10 (s, 1H),7.18-7.23 (m, 2H), 7.43-7.52 (m, 4H); MS (DCI/NH₃) m/z 385.0 (M+H)⁺.

Example 27 tert-butyl4-[2-({4-[(difluoromethyl)thio]phenyl}amino)-2-oxoethyl]phenylcarbamate

The product was prepared from2-(4-(tert-butoxycarbonylamino)phenyl)acetic acid (2.76 g) according toMethod C: ¹H NMR (300 MHz, CDCl₃) δ ppm 1.53 (s, 9H), 3.68-3.71 (m, 2H),6.52 (s, 1H), 6.75 (t, J=57.0 Hz, 1H), 7.09 (s, 1H), 7.21-7.27 (m, 2H),7.35-7.42 (m, 2H), 7.42-7.52 (m, 4H); MS (DCI/NH₃) m/z 426.1 (M+NH₄)⁺.

Example 28 2-(4-aminophenyl)-N-{4-[(difluoromethyl)thio]phenyl}acetamide

Tert-butyl4-[2-({4-[(difluoromethyl)thio]phenyl}amino)-2-oxoethyl]phenylcarbamate(Example 27) (3.63 g) was stirred in 25% trifluoroaceticacid/dichloromethane at room temperature for 45 minutes. The mixture wasdiluted with 1,2-dichloroethane (25 mL) and evaporated down twice. Theresidue was recrystallized from ethyl acetate (15 mL) and hexane (15mL). The product was obtained as the 1:1 trifluoroacetate salt,2-(4-aminophenyl)-N-(4-(difluoromethylthio)phenyl)acetamide2,2,2-trifluoroacetate: ¹H NMR (300 MHz, DMSO-d₆) δ ppm 3.45-4.13 (m,2H) 3.62 (s, 2H) 7.03 (d, J=8.5 Hz, 2H) 7.38 (t, J=56.1 Hz, 1H) 7.28 (d,J=8.5 Hz, 2H) 7.47-7.55 (m, 2H) 7.64-7.72 (m, 2H) 10.36 (s, 1H) MS(DCI/NH₃) m/z 309.0 (M+H)⁺; Anal. C₁₅H₁₄F₂N₂OS C₂HF₃O₂: C, H, N.

Example 29N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(methylamino)phenyl]acetamide

Formic acid (0.35 mL, 9.1 mmol) was added to acetic anhydride (0.69 mL,7.3 mmol) at 0° C. The mixture was warmed to 60° C. for 2 hours, thencooled to 0° C. To the anhydride mixture was added a solution of methyl2-(4-aminophenyl)acetate (0.95 g, 5.7 mmol) in tetrahydrofuran (14 mL).The mixture was warmed to room temperature and stirred for 18 hours. Thereaction mixture was neutralized with saturated aqueous sodiumbicarbonate and diluted with ether. The ether layer was separated, driedover sodium sulfate, and concentrated. The residue was filtered throughsilica (1% methanol/dichloromethane eluent) to give methyl2-(4-formamidophenyl)acetate (0.8 g, 4.14 mmol, 56% yield). The productwas dissolved in tetrahydrofuran (14 mL) and cooled to 0° C. Boranemethyl sulfide complex (0.393 mL, 4.14 mmol) was added. After 15minutes, the mixture was warmed to room temperature for 30 minutes. Themixture was cooled to 0° C. again, and 5 mL of methanol was added. Themixture was concentrated and the residue was purified by silicachromatography (10% ethyl acetate/hexane eluent) to give methyl2-(4-(methylamino) phenyl)acetate: ¹H NMR (300 MHz, CDCl₃) δ ppm 2.82(s, 3H), 3.51 (s, 2H), 3.67 (s, 3H), 6.51-6.61 (m, 2H), 6.99-7.15 (m,2H); MS (ESI+) m/z 179.9 (M+H)⁺.

Methyl 2-(4-(methylamino)phenyl)acetate (721 mg, 4.0 mmol) was dissolvedin tetrahydrofuran (10 mL) and treated with a 1 M aqueous solution oflithium hydroxide (4.4 mmol). After 3 hours, the mixture was acidifiedwith 1 N aqueous hydrochloric acid and concentrated to removetetrahydrofuran. The aqueous phase was extracted with dichloromethane(2×20 mL) and the organic layer was dried and concentrated to give2-(4-(methylamino)phenyl)acetic acid (309 mg, 1.87 mmol).

The product was prepared from 2-(4-(methylamino)phenyl)acetic acid (54mg) according to Method C: ¹H NMR (300 MHz, DMSO-d₆) δ ppm 2.64 (d,J=5.2 Hz, 3H), 3.46 (s, 2H), 5.50 (q, J=4.9 Hz, 1H), 6.43-6.53 (m, 2H),6.99-7.09 (m, 2H), 7.37 (t, J=56.1 Hz, 1H), 7.45-7.54 (m, 2H), 7.63-7.73(m, 2H), 10.24 (s, 1H); MS (DCI/NH₃) m/z 322.9 (M+H)⁺.

Example 30N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(dimethylamino)phenyl]acetamide

The product was prepared from 2-(4-(dimethylamino)phenyl)acetic acid (54mg) according to Method C: ¹H NMR (300 MHz, DMSO-d₆) δ ppm 2.85 (s, 6H),3.50 (s, 2H), 6.64-6.71 (m, 2H), 7.09-7.16 (m, 2H), 7.37 (t, J=56.1 Hz,1H), 7.45-7.54 (m, 2H), 7.63-7.71 (m, 2H), 10.27 (s, 1H); MS (DCI/NH₃)m/z 337.1 (M+H)⁺.

Example 312-[4-(dimethylamino)phenyl]-N-{4-[(trifluoromethyl)thio]phenyl}acetamide

The product was prepared from 2-(4-(dimethylamino)phenyl)acetic acid (23mg) and 4-trifluoromethylthioaniline (28 mg) according to Method C: ¹HNMR (300 MHz, CDCl₃) δ ppm 2.98 (s, 6H), 3.66 (s, 2H), 6.72-6.78 (m,2H), 7.14-7.19 (m, 2H), 7.19 (s, 1H), 7.43-7.51 (m, 2H), 7.52-7.57 (m,2H); MS (ESI-pos) m/z 355.0 (M+H)⁺.

Example 32N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(isobutylamino)phenyl]acetamide

The product was prepared using isobutyraldehyde according to Method D:¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.92 (d, J=6.7 Hz, 6H), 1.69-1.89 (m,1H), 2.83 (dd, J=5.8, 6.9 Hz, 2H), 3.48 (s, 2H), 6.54-6.66 (m, 2H),7.03-7.12 (m, 2H), 7.33 (t, J=55.8 Hz, 1H), 7.48-7.56 (m, 2H), 7.63-7.72(m, 2H); MS (ESI+) m/z 365.0 (M+H)⁺.

Example 332-{4-[(cyclohexylmethyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using cyclohexanecarbaldehyde according toMethod D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.92 (m, 2H), 1.16 (m, 2H),1.19 (m, 1H), 1.47-1.58 (m, J=18.0, 14.5, 7.0, 3.8 Hz, 1H), 1.62 (m,1H), 1.68 (m, 2H), 1.76 (m, 2H), 2.87 (ABX, 2H), 3.49 (s, 2H), 6.63-6.69(m, 2H), 7.07-7.13 (m, 2H), 7.33 (t, J=56.1 Hz, 1H), 7.48-7.55 (m, 2H),7.64-7.70 (m, 2H); MS (ESI−) m/z 403.1 (M−H)⁻.

Example 34N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(2-methoxybenzyl)amino]phenyl}acetamide

The product was prepared using 2-methoxybenzaldehyde according to MethodD: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.47 (s, 2H), 3.82 (s, 3H), 4.22 (s,2H), 6.58 (d, J=8.2 Hz, 2H), 6.88 (t, J=7.3 Hz, 1H), 6.99 (d, J=8.5 Hz,1H), 7.03-7.09 (m, 2H), 7.20-7.24 (m, 2H), 7.33 (t, J=56.7 Hz, 1H),7.48-7.54 (m, 2H), 7.62-7.69 (m, 2H), 10.34 (s, 1H); MS (ESI−) m/z 427.1(M−H)⁻.

Example 352-{4-[(4-chlorobenzyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using 4-chlorobenzaldehyde according to MethodD: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.44 (s, 2H), 4.24 (s, 2H), 6.47-6.55(m, 2H), 6.98-7.06 (m, 2H), 7.33 (t, J=56.0 Hz, 1H), 7.34-7.38 (m, 4H),7.48-7.53 (m, 2H), 7.63-7.68 (m, 2H), 10.32 (s, 1H); MS (ESI−) m/z 431.0(M−H)⁻.

Example 362-{4-[(2-chlorobenzyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using 2-chlorobenzaldehyde according to MethodD: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.45 (s, 2H), 4.32 (s, 2H), 6.44-6.53(m, 2H), 7.00-7.07 (m, 2H), 7.24-7.29 (m, 2H), 7.33 (t, J=56.6 Hz, 1H),7.38 (dd, J=6.9, 5.2, Hz, 1H), 7.44 (d, J=6.6, 2.7 Hz, 1H), 7.49-7.54(m, 2H), 7.63-7.69 (m, 2H), 10.33 (s, 1H); MS (ESI−) m/z 431.0 (M−H)⁻.

Example 37N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(3-methoxybenzyl)amino]phenyl}acetamide

The product was prepared using 3-methoxybenzaldehyde according to MethodD: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.46 (s, 2H), 3.71 (s, 3H), 4.24 (s,2H), 6.56-6.61 (m, 2H), 6.79 (dd, J=8.1, 2.3 Hz, 1H), 6.91 (dd, J=2.4,1.7 Hz, 1H), 6.93 (dd, J=8.0, 1.6 Hz, 1H), 7.01-7.06 (m, 2H), 7.22 (t,J=7.6 Hz, 1H), 7.33 (t, J=56.7 Hz, 1H), 7.48-7.53 (m, 2H), 7.64-7.68 (m,2H), 10.33 (s, 1H); MS (ESI−) m/z 427.1 (M−H)⁻.

Example 38N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(3-methylbutyl)amino]phenyl}acetamide

The product was prepared using 3-methylbutanal according to Method D: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 0.89 (d, J=6.4 Hz, 6H), 1.44 (m, 2H), 1.66(septet, J=6.6 Hz, 1H), 3.04 (dd, J=7.8, 7.4 Hz, 2H), 3.52 (s, 2H),6.70-6.77 (m, 2H), 7.11-7.18 (m, 2H), 7.33 (t, J=56.0 Hz, 1H), 7.50-7.54(m, 2H), 7.64-7.69 (m, 2H), 10.37 (s, 1H); MS (ESI+) m/z 379.1 (M+H)⁺.

Example 39N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(quinolin-4-ylmethyl)amino]phenyl}acetamide

The product was prepared as the trifluoroacetate salt usingquinoline-4-carbaldehyde according to Method D: ¹H NMR (500 MHz,DMSO-d₆) δ ppm 3.47 (s, 2H), 4.99 (s, 2H), 6.55-6.60 (m, 2H), 7.05-7.09(m, 2H), 7.33 (t, J=56.0 Hz, 1H), 7.49-7.53 (m, 2H), 7.53 (m, 1H),7.64-7.68 (m, 2H), 7.76 (d, J=5.2 Hz, 1H), 7.91 (ddd, J=8.5, 7.2, 1.1Hz, 1H), 8.06 (ddd, J=8.4, 7.0, 1.1 Hz, 1H), 8.21 (d, J=7.9 Hz, 1H),9.02 (d, J=5.2 Hz, 1H), 10.36 (s, 1H); MS (ESI−) m/z 448.1 (M−H)⁻.

Example 40N-{4-[(difluoromethyl)thio]phenyl}-2-(4-{[(5-ethyl-2-furyl)methyl]amino}phenyl)acetamide

The product was prepared using 2-(5-ethylfuran-2-yl)acetaldehydeaccording to Method D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.13 (t, J=7.5Hz, 3H), 2.56 (q, J=7.6 Hz, 2H), 3.50 (s, 2H), 4.21 (s, 2H), 5.98 (d,J=3.1 Hz, 1H), 6.18 (d, J=3.1 Hz, 1H), 6.66-6.74 (m, 2H), 7.07-7.14 (m,2H), 7.33 (t, J=56.1 Hz, 1H), 7.49-7.54 (m, 2H), 7.63-7.70 (m, 2H), 9.47(s, 1H); MS (ESI−) m/z 415.1 (M−H)⁻.

Example 41N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(tetrahydro-2H-pyran-4-ylamino)phenyl]acetamide

The product was prepared using dihydro-2H-pyran-4(3H)-one according toMethod D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.56 (dq, J=11.7, 4.3 Hz, 2H),1.81 (dd, J=12.5, 2.1 Hz, 2H), 3.34 (td, J=11.7, 1.5 Hz, 2H), 3.60 (tt,J=11.2, 11.2, 4.2, 4.0 Hz, 1H), 3.67 (s, 2H), 3.90 (dd, J=11.7, 2.6 Hz,2H), 7.14-7.19 (m, 2H), 7.34 (t, J=56.2 Hz, 1H), 7.36-7.40 (m, 2H),7.51-7.55 (m, 2H), 7.66-7.70 (m, 2H), 10.49 (s, 1H); MS (ESI−) m/z 391.1(M−H)⁻.

Example 42N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(4-phenoxybenzyl)amino]phenyl}acetamide

The product was prepared using 4-phenoxybenzaldehyde according to MethodD: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.48 (s, 2H), 4.26 (s, 2H), 6.56-6.68(m, 2H), 6.92-6.99 (m, 4H), 7.04-7.10 (m, 2H), 7.13 (t, J=7.5 Hz, 1H),7.33 (t, J=56.0 Hz, 3H), 7.35-7.41 (m, 4H), 7.49-7.54 (m, 2H), 7.64-7.68(m, 2H); MS (ESI−) m/z 489.1 (M−H)⁻.

Example 432-{4-[(cyclopropylmethyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using cyclopropanecarbaldehyde according toMethod D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.32 (ddd, J=6.0, 4.6, 4.4 Hz,2H), 0.56 (ddd, J=8.1, 6.6, 4.5 Hz, 2H), 1.02 (m, 1H), 3.13 (d, J=7.3Hz, 2H), 3.67 (s, 2H), 7.20-7.25 (m, 2H), 7.34 (t, J=56.0 Hz, 1H),7.36-7.41 (m, 2H), 7.51-7.56 (m, 2H), 7.64-7.71 (m, 2H), 10.49 (s, 1H);MS (ESI+) m/z 363.0 (M+H)⁺.

Example 442-(4-{[(4-bromothien-2-yl)methyl]amino}phenyl)-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using 4-bromothiophene-2-carbaldehyde accordingto Method D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.46 (s, 2H), 4.42 (s, 2H),6.50-6.63 (m, 2H), 7.03 (d, J=1.2 Hz, 1H), 7.03-7.07 (m, 2H), 7.33 (t,J=56.1 Hz, 1H), 7.42 (d, J=1.5 Hz, 1H), 7.49-7.54 (m, 2H), 7.64-7.69 (m,2H), 10.34 (s, 1H); MS (ESI−) m/z 482.9 (M−H)⁻.

Example 45N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(quinolin-2-ylmethyl)amino]phenyl}acetamide

The product was prepared as the trifluoroacetate salt usingquinoline-2-carbaldehyde according to Method D: ¹H NMR (500 MHz,DMSO-d₆) δ ppm 3.46 (s, 2H), 4.69 (s, 2H), 6.59-6.68 (m, 2H), 7.02-7.09(m, 2H), 7.32 (t, J=56.0 Hz, 1H), 7.48-7.52 (m, 2H), 7.62-7.66 (m, 2H),7.73 (m, 1H), 7.74 (d, J=8.5 Hz, 1H), 7.93 (m, 1H), 8.10 (d, J=8.2 Hz,1H), 8.13 (m, 1H), 8.65 (d, J=8.5 Hz, 1H), 10.34 (s, 1H); MS (ESI−) m/z448.1 (M−H)⁻.

Example 462-(4-{[(1-acetyl-1H-indol-3-yl)methyl]amino}phenyl)-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using 1-acetyl-1H-indole-3-carbaldehydeaccording to Method D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.61 (s, 3H),3.48 (s, 2H), 4.40 (s, 2H), 6.68-6.78 (m, 2H), 7.06-7.14 (m, 2H), 7.28(t, J=7.5 Hz, 1H), 7.33 (t, J=56.4 Hz, 1H), 7.34 (t, J=7.6 Hz, 1H),7.47-7.54 (m, 2H), 7.63-7.70 (m, 2H), 7.72 (d, J=7.6 Hz, 1H), 7.80 (s,1H), 8.30 (d, J=8.2 Hz, 1H); MS (ESI−) m/z 478.1 (M−H)⁻.

Example 472-[4-(cyclohexylamino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using cyclohexanone according to Method D: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 1.13 (qt, J=12.3, 2.1 Hz, 1H), 1.25 (qt,J=12.8, 2.8 Hz, 2H), 1.32 (m, 2H), 1.60 (dt, J=12.8, 2.0 Hz, 1H), 1.75(dt, J=12.8, 2.2 Hz, 2H), 1.88 (ddd, J=11.0, 3.5, 1.9 Hz, 2H), 3.35 (tt,J=10.8, 3.5 Hz, 1H), 3.71 (s, 2H), 7.26-7.32 (m, 2H), 7.34 (t, J=56.0Hz, 1H), 7.39-7.48 (m, 2H), 7.48-7.57 (m, 2H), 7.63-7.74 (m, 2H), 10.52(s, 1H); MS (ESI−) m/z 389.1 (M−H)⁻.

Example 482-[4-({[5-(2-chlorophenyl)-2-furyl]methyl}amino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using 5-(2-chlorophenyl)furan-2-carbaldehydeaccording to Method D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.48 (s, 2H),4.32 (s, 2H), 6.45 (d, J=3.4 Hz, 1H), 6.65-6.69 (m, 2H), 7.05 (d, J=3.4Hz, 1H), 7.06-7.11 (m, 2H), 7.30 (td, J=7.7, 1.7 Hz, 1H), 7.33 (t,J=56.0 Hz, 1H), 7.41 (td, J=7.6, 1.2 Hz, 1H), 7.50-7.54 (m, 2H), 7.52(m, 1H), 7.65-7.69 (m, 2H), 7.80 (dd, J=7.9, 1.5 Hz, 1H), 10.34 (s, 1H);MS (ESI−) m/z 497.1 (M−H)⁻.

Example 49N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(4-methoxybenzyl)amino]phenyl}acetamide

The product was prepared using 4-methoxybenzaldehyde according to MethodD: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.45 (s, 2H), 3.71 (s, 3H), 4.18 (s,2H), 6.53-6.61 (m, 2H), 6.78-6.93 (m, 2H), 6.98-7.09 (m, 2H), 7.25-7.30(m, 2H), 7.33 (t, J=56.0 Hz, 1H), 7.47-7.56 (m, 2H), 7.60-7.72 (m, 2H),10.33 (s, 1H); MS (ESI−) m/z 427.1 (M−H)⁻.

Example 502-[4-(cyclopentylamino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using cyclopentanone according to Method D: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 1.52-1.65 (m, 4H), 1.72-1.75 (m, 2H),1.83-1.94 (m, 2H), 3.69 (s, 2H), 3.85 (pentet, J=7.0 Hz, 1H), 7.20-7.27(m, 2H), 7.34 (t, J=56.0 Hz, 1H), 7.39-7.43 (m, 2H), 7.49-7.60 (m, 2H),7.65-7.72 (m, 2H), 10.51 (s, 1H); MS (ESI+) m/z 377.2 (M+H)⁺.

Example 512-{4-[(3-chlorobenzyl)amino]phenyl}-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using 3-chlorobenzaldehyde according to MethodD: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.45 (s, 2H), 4.27 (s, 2H), 6.44-6.59(m, 2H), 6.98-7.09 (m, 2H), 7.27 (ddd, J=7.5, 1.8, 1.7 Hz, 1H), 7.31(ddd, J=7.5, 1.8, 1.7 Hz, 1H), 7.33 (t, J=56.1 Hz, 1H), 7.34 (t, J=7.7Hz, 1H), 7.38 (dd, J=1.9, 1.7 Hz, 1H), 7.47-7.58 (m, 2H), 7.62-7.71 (m,2H); MS (ESI−) m/z 431.0 (M−H)⁻.

Example 522-[4-(cyclobutylamino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using cyclobutanone according to Method D: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 1.77 (m, 2H), 2.04 (m, 2H), 2.22 (m, 2H),3.62 (s, 2H), 3.94 (dt, J=15.6, 7.9 Hz, 1H), 6.97-7.04 (m, 2H), 7.34 (t,J=56.0 Hz, 1H), 7.29-7.33 (m, 2H), 7.50-7.57 (m, 2H), 7.65-7.73 (m, 2H),10.46 (s, 1H); MS (ESI−) m/z 361.1 (M−H)⁻.

Example 532-[4-(cycloheptylamino)phenyl]-N-{4-[(difluoromethyl)thio]phenyl}acetamide

The product was prepared using cycloheptanone according to Method D: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 1.41 (m, 2H), 1.46-1.60 (m, 6H), 1.68 (m,2H), 1.91 (m, 2H), 3.55 (ddd, J=13.9, 9.5, 4.1 Hz, 1H), 3.71 (s, 2H),7.25-7.32 (m, 2H), 7.34 (s, 1H), 7.41-7.48 (m, 2H), 7.51-7.57 (m, 2H),7.66-7.72 (m, 2H); MS (ESI−) m/z 403.1 (M−H)⁻.

Example 54N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(2-methylbutyl)amino]phenyl}acetamide

The product was prepared using 2-methylbutanal according to Method D: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 0.87 (t, J=7.5 Hz, 3H), 0.93 (d, J=6.7 Hz,3H), 1.16 (dqd, J=7.6, 7.5, 6.3, 1H), 1.41-1.51 (dqd, J=7.6, 7.5, 6.0Hz, 1H), 1.65 (ddqdd, J=7.3, 7.2, 6.7, 6.3, 6.0 Hz, 1H), 2.89 (dd,J=12.5, 7.3 Hz, 1H), 3.02 (dd, J=12.7, 6.3 Hz, 1H), 3.55 (s, 2H),6.80-6.89 (m, 2H), 7.18-7.22 (m, 2H), 7.33 (t, J=56.0 Hz, 1H), 7.50-7.55(m, 2H), 7.62-7.71 (m, 2H), 10.40 (s, 1H); MS (ESI+) m/z 379.0 (M+H)⁺.

Example 55N-{4-[(difluoromethyl)thio]phenyl}-2-(4-{[(5-methylthien-2-yl)methyl]amino}phenyl)acetamide

The product was prepared using 5-methylthiophene-2-carbaldehydeaccording to Method D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.35 (s, 3H),3.46 (s, 2H), 4.34 (s, 2H), 6.59-6.62 (m, 2H), 6.61 (d, J=3.4 Hz, 1H),6.80 (d, J=3.4 Hz, 1H), 7.02-7.08 (m, 2H), 7.33 (t, J=56.0 Hz, 1H),7.48-7.55 (m, 2H), 7.63-7.71 (m, 2H); MS (ESI−) m/z 417.0 (M−H)⁻.

Example 56N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(2-naphthylmethyl)amino]phenyl}acetamide

The product was prepared using 2-naphthaldehyde according to Method D:¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.45 (s, 2H), 4.44 (s, 2H), 6.60-6.67(m, 2H), 7.01-7.08 (m, 2H), 7.32 (t, J=56.0 Hz, 1H), 7.46-7.49 (m, 2H),7.49-7.52 (m, 2H), 7.53 (dd, J=6.5, 1.5 Hz, 1H), 7.62-7.67 (m, 2H), 7.84(dd, J=5.3, 4.4 Hz, 2H), 7.86-7.89 (m, 2H), 10.32 (s, 1H); MS (ESI−) m/z447.1 (M−H)⁻.

Example 57N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(3,3,5,5-tetramethylcyclohexyl)amino]phenyl}acetamide

The product was prepared using 3,3,5,5-tetramethylcyclohexanoneaccording to Method D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.91 (s, 6H),0.99 (s, 6H), 1.08 (d, J=14.0 Hz, 1H), 1.13 (t, J=12.2 Hz, 2H), 1.27 (d,J=13.7 Hz, 1H), 1.65 (d, J=11.6 Hz, 2H), 3.67 (tt, J=11.9, 3.4 Hz, 1H),3.70 (s, 2H), 7.34 (t, J=56.0 Hz, 1H), 7.24-7.30 (m, 2H), 7.40-7.44 (m,2H), 7.50-7.56 (m, 2H), 7.67-7.71 (m, 2H), 10.52 (s, 1H); MS (ESI+) m/z447.1 (M+H)⁺.

Example 58N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(thien-2-ylmethyl)amino]phenyl}acetamide

The product was prepared using thiophene-2-carbaldehyde according toMethod D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.46 (s, 2H), 4.43 (s, 2H),6.56-6.66 (m, 2H), 6.96 (dd, J=5.0, 3.5 Hz, 1H), 7.03 (dd, J=3.4, 0.9Hz, 1H), 7.33 (dd, J=5.2, 1.2 Hz, 1H), 7.33 (t, J=56.0 Hz, 1H),7.47-7.57 (m, 2H), 7.62-7.70 (m, 2H); MS (ESI−) m/z 403.0 (M−H)⁻.

Example 59N-{4-[(difluoromethyl)thio]phenyl}-2-[4-(neopentylamino)phenyl]acetamide

The product was prepared using pivalaldehyde according to Method D: ¹HNMR (500 MHz, DMSO-d₆) δ ppm 0.97 (s, 9H), 2.88 (s, 2H), 3.53 (s, 2H),6.79-6.88 (m, 2H), 7.10-7.19 (m, 2H), 7.33 (t, J=56.0 Hz, 1H), 7.48-7.57(m, 2H), 7.63-7.73 (m, 2H), 10.38 (s, 1H); MS (ESI+) m/z 379.0 (M+H)⁺.

Example 60N-{4-[(difluoromethyl)thio]phenyl}-2-(4-{[4-(trifluoromethyl)cyclohexyl]amino}phenyl)acetamide

The product was prepared using 4-(trifluoromethyl)cyclohexanoneaccording to Method D: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.30-1.42 (m,4H), 1.94 (m, 2H), 2.00 (m, 2H), 2.27 (m, 1H), 3.37 (m, 1H), 3.64 (s,2H), 7.05-7.14 (m, 2H), 7.34 (t, J=56.0 Hz, 1H), 7.32-7.38 (m, 2H),7.50-7.56 (m, 2H), 7.64-7.71 (m, 2H); MS (ESI+) m/z 459.1 (M+H)⁺.

Example 61N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(2,2,2-trifluoroethyl)amino]phenyl}acetamide

The product was prepared using trifluoroacetaldehyde according to MethodD: ¹H NMR (500 MHz, DMS-d₆) δ ppm 3.49 (s, 2H), 3.86 (q, J=9.7 Hz, 2H),6.65-6.71 (m, 2H), 7.06-7.12 (m, 2H), 7.33 (t, J=56.0 Hz, 1H), 7.50-7.54(m, 2H), 7.63-7.70 (m, 2H), 10.36 (s, 1H); MS (ESI−) m/z 389.0 (M−H)⁻.

Example 62N-{4-[(difluoromethyl)thio]phenyl}-2-{4-[(3-phenylcyclohexyl)amino]phenyl}acetamide

The product was prepared using 3-phenylcyclohexanone according to MethodD: ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.55 (qd, J=11.9, 2.1 Hz, 1H),1.59-1.69 (m, 2H), 1.72-1.92 (m, 6H), 2.99 (tt, J=11.1, 2.1 Hz, 1H),3.60 (s, 2H), 7.00-7.07 (m, 2H), 7.19 (t, J=7.3 Hz, 1H), 7.21-7.26 (m,2H), 7.26-7.33 (m, 4H), 7.33 (t, J=56.0 Hz, 1H), 7.49-7.56 (m, 2H),7.64-7.70 (m, 2H); MS (ESI−) m/z 465.2 (M−H)⁻.

DETERMINATION OF BIOLOGICAL ACTIVITY

To determine the effectiveness as allosteric modulators, the compoundsof the invention were evaluated according to the following assays usingXenopus oocytes, cells or cell lines expressing endogenous orrecombinant α7 NNRs.

The assays include (i) Xenopus oocytes injected with α7 NNR cRNA or cDNAand evaluation of compound effects on current responses evoked byacetylcholine or another agonist (ii) IMR-32 cells endogenouslyexpressing α7 NNRs and measuring Ca²⁺ flux or membrane potential changesutilizing the fluorescence-imaging plate reader (FLIPR)-based assays and(iii) measurement of phospho-ERK activity using western blot assays.

To determine the effectiveness of compounds of formula (I) in reducingpain, the compounds of the invention were evaluated in theformalin-induced persistent nociceptive behavior assay.

A sensory gating (N40) assay in DBA/2 mice was used to determine whetheror not compounds of the invention enhance the ability of the testanimals to focus on important stimuli and ignore irrelevant backgroundnoise.

Binding to sigma receptors (σ1 and σ2) were used as a selectivity assay.

(i) Two-Electrode Voltage-Clamp in Xenopus laevis Oocytes

X. laevis oocytes were prepared for electrophysiological experiments asdescribed in the literature (see for example, Briggs, C. A. et al,Neuropharmacology, 1995, 34: 583-590; Briggs, C. A. et al.,Neuropharmacology, 1998, 37: 1095-1102, which are incorporated herein byreference). In brief, three to four lobes from ovaries of female adultX. laevis frogs were removed and defolliculated after treatment withcollagenase type 1A (2 mg/mL; Sigma) prepared in low-Ca²⁺ Barth'ssolution (90 mM NaCl, 1.0 mM KCl, 0.66 mM NaNO₃, 2.4 mM NaHCO₃, 10 mMHEPES, 2.5 mM sodium pyruvate, 0.82 mM MgCl₂, and 0.5% (v/v)penicillin-streptomycin solution, pH=7.55, Sigma) for about 1.5 hours toabout 2 hours at about 18° C. under constant agitation to obtainisolated oocytes.

The isolated oocytes were injected with about 4 ng to about 6 ng ofhuman α7 NNR cRNA, kept at about 18° C. in a humidified incubator inmodified Barth's solution (90 mM NaCl, 1.0 mM KCl, 0.66 mM NaNO₃, 2.4 mMNaHCO₃, 10 mM HEPES, 2.5 mM sodium pyruvate, 0.74 mM CaCl₂, 0.82 mMMgCl₂, 0.5% (v/v) penicillin-streptomycin solution, pH 7.55) and usedabout 2 to 7 days after injection. Responses were measured bytwo-electrode voltage clamp using a Parallel Oocyte ElectrophysiologyTest Station (Abbott, Abbott Park, Ill.) (see for example, Trumbull, J.D., et al., Receptors Channels, 2003, 9: 19-28, which is incorporatedherein by reference). During recordings, the oocytes were bathed inBa²⁺—OR2 solution (90 mM NaCl, 2.5 mM KCl, 2.5 mM BaCl₂, 1.0 mM MgCl₂,5.0 mM HEPES, and 0.0005 mM atropine, pH 7.4) to prevent activation ofCa²⁺-dependent currents and held at −60 mV at room temperature (about20° C.). Test compounds were pre-applied alone for ˜60 seconds andsubsequently in the presence of 0.1 mM acetylcholine (ACh) as theagonist. The data were expressed as percentage potentiation of theacetylcholine-evoked current normalized to the response of reference PAM(at 10 μM,N′-[(2Z)-3-(2,2-difluoro-1,3-benzodioxol-5-yl)-5-methyl-1,3-thiazol-2(3H)-ylidene]-N,N-dimethylurea)taken as 100% and the response to submaximum ACh without any PAM as 0%.

FIG. 1 shows the concentration-response relationship for Example 17 inpotentiating 0.1 mM ACh-evoked α7 currents in oocytes. In this graph,the EC₅₀ value is 0.18 μM and the degree of normalized potentiation is94%. FIG. 2 shows the concentration-response relationship for Example 12in potentiating 0.1 mM ACh-evoked α7 currents in oocytes. In this graph,the EC₅₀ value is 3.7 μM and the degree of potentiation is 61%.

(ii) High-Throughput Calcium Flux Assays Using Cells ExpressingEndogenous α7 NNRs

Since allosteric modulators affect the kinetics of channel function andthus affect calcium dynamics, it is demonstrated that novel modulatorscan be identified when assays are conducted in the presence of aselective agonist, and conversely, novel agonists can be identified whenscreened or tested in the presence an allosteric modulator. As such,PAMs and NNR agonists can be identified by using IMR-32 cells thatendogenously express various nicotinic receptors including α7 NNRs. Itis contemplated that such an assay can be utilized with a number of celllines that express α7 NNR subunits, and conventionally not amenable toα7 nicotinic compound screening. Accordingly, allosteric modulatorcompounds described herein can be identified using a fluorescence-basedthroughput functional assay using cell lines such as IMR-32neuroblastoma or primary dissociated neurons.

Although cell types such as IMR-32 neuroblastoma and neurons are knownto contain several nicotinic receptor subunits, α7 selective agonists inthe present assay selectively stimulate calcium responses only in thepresence of PAMs. Any suitable selective α7 agonist can be used.Selective α7 agonists from a range of structural types may be used suchas those described in the literature including PNU-282987, SSR180711Aand AR-R17779, and others described in earlier patent applications, suchas 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole(see for example, US 20050065178),5-[6-(5-methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-1H-indole(see for example, US 20050065178),3-[6-(1H-indol-5-yl)-pyradazin-3-yloxy]-1-aza-bicyclo[2.2.2]octane (seefor example, US 2005/0137204 and US 2005/0245531), and4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-1,4-diaza-bicyclo[3.2.2]nonane (seefor example, WO 2004/029053).

IMR-32 neuroblastoma cells (ATCC) were grown to confluency in 162 cm²tissue culture flasks in minimum essential media supplemented with 10%fetal bovine serum and 1 mM sodium pyruvate, 0.1 mM non-essential aminoacids and 1% antibiotic-antimycotic. The cells were then dissociatedusing cell dissociation buffer and 100 μL of 3.5×10⁵ cells/mL cellsuspension was plated (about 75,000-100,000 cells/well) into black 96well plates precoated with poly-D-lysine with a clear bottom andmaintained for 24-48 hours in a tissue culture incubator at 37° C. underan atmosphere of 5% CO₂: 95% air. Other clonal cell lines or dissociatedprimary cortical neurons that express endogenous α7 nicotinic receptorsmay also be used in this assay.

Calcium flux was measured using a calcium-3 assay kit (MolecularDevices, Sunnyvale, Calif.) or Fluo-4 (Invitrogen, Carlsbad, Calif.). Astock solution of the dye was prepared by dissolving each vial suppliedby the vendor in Hank's balanced salt solution buffer (HBSS) containing10 or 20 mM HEPES. The stock solution was diluted 1:20 using the samebuffer before use. The growth media was removed from the cells andloaded with 100 μL of the dye and incubated at room temperature for oneto three hours. Fluorescence measurements were read simultaneously fromall the wells by a Fluorometric Imaging Plate Reader (FLIPR) at anexcitation wavelength of 480 nm and an emission wavelength of 520 nm.Baseline fluorescence was measured for the first 10 seconds at which 50μL of 3× concentrations of modulator/test compounds were added to thecell plate and incubated for three to five minutes. The fluorescenceintensity was captured every second for the first 1 minute followed byevery 2-5 seconds for an additional two to four minutes. This procedurewas followed by 50 μL of 4× concentration of agonist and readings weretaken for a period of three to five minutes as described above.

The assay can also be adapted to other formats such as 384- or 1536-wellformats. The concentration dependence of changes in fluorescenceresponses was fitted by nonlinear regression analysis (GraphPad Prism,San Diego, Calif.) or Assay Explorer (Elsevier MDL, San Ramon, Calif.)to obtain EC₅₀ values. Agonist alone did not evoke any responses.However, in the presence of an allosteric modulator, the agonistelicited a concentration dependent increase in calcium response. The α7NNR selective antagonist, methyllycaconitine (MLA), abolished theresponse demonstrating that the effects are mediated via α7 NNR.

PAMs were identified by measuring fluorescence changes to intracellularcalcium in a fluorometric plate reader in the presence of a selective α7NNR agonist using cells natively expressing α7 NNRs. Compounds with PAMactivity evoked a calcium fluorescence response in the IMR-32neuroblastoma cell line, a cell line that expresses endogenous α7 NNRswhen the assay is conducted in presence of an α7 NNR agonist. Theagonist alone did not evoke a calcium response. However, when theagonist and the modulator were co-applied together, calcium responseswere triggered. In the example above,4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-1,4-diaza-bicyclo[3.2.2]nonane (seefor example, WO 2004/029053) was used as an agonist at 1 μM in thepresence of varying concentrations of compounds of the invention. TheEC₅₀ values of PAM compounds as determined in this assay typically canrange from 1 nM to >30 μM. Other α7 NNR agonists including2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole(published in US 20050065178),5-[6-(5-methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-1H-indole(published in US 20050065178), various quinuclidine derivatives (see forexample, US 2005/0137204 and US 2005/0245531) and PNU-282987 (see forexample, Hajos, M., et al., J Pharmacol. Exp Ther., 2005, 312: 1213-22)also are suitable for the assay. Likewise, primary neurons and otherclonal cell lines that natively express α7 NNRs may also be utilized.Other fluorescence measurements such as those monitoring changes inmembrane potential also are suitable for the assay.

(iii) High-Throughput ERK Phosphorylation Assays Using Cells ExpressingEndogenous α7 NNRs

Rat pheochromocytoma (PC-12) cells (ATCC, Manassas, Va.) were culturedand maintained in F-12K media supplemented with 15% horse serum, 2.5%fetal calf serum, and 2 mM L-glutamine in poly-D lysine coated dishes at37° C. and 5% CO₂. Cells were plated in black-walled clear bottom96-well Biocoat™ plates coated with poly-D-lysine (BD Biosciences,Bedford, Mass.) and grown for 2-3 days. Afterward, the culture media isreplaced with serum-free media to starve cells overnight. On the day ofthe assay, cell media was removed and cells (60-80% confluent) weretreated with agonist and/or modulator in Dulbecco's phosphate buffersaline (D-PBS) (with Ca²⁺, Mg²⁺, and 1 mg/mL D-glucose), as indicated inthe result section.

PC-12 cells are treated for 10 minutes at 37° C. with test PAM compoundsand then challenged with a selective α7 NNR agonist for 5 minutes at 37°C. in a final volume of 100 μL/well, unless otherwise indicated. Aftertreatment, media was discarded and adherent cells were immediately fixedin the presence of 150 μL/well of 3.7% formaldehyde/phosphate-bufferedsaline for 30-60 minutes at room temperature. Cells were then washed (4times/5 minutes) and permeabilized with 200 μL/well of 0.1% TritonX-100/PBS. Permeabilized cells were blocked using the Odyssey® blockingbuffer (100 μL/well) and plates were rocked overnight at 4° C. Bothanti-total ERK (rabbit) and anti-phospho ERK (mouse) antibodies werediluted to 1/1000 and 1/500, respectively, in Odyssey® blocking bufferand added together at 50 μL/well for 2-3 hours at room temperature.Polyclonal rabbit anti-ERK1/2 and monoclonal mouse anti-phospho-ERK 1/2were purchased from Sigma-Aldrich (St. Louis, Mo.). The plates werewashed 4 times with 0.1% Tween 20/PBS (200 uL/well), and incubated withsecondary antibodies (1/1000 dilution) in blocking buffer supplementedwith 0.2% Tween for 1 hour. Alexa Fluor® 680-labeled goat anti-rabbitantibodies were added to recognize total ERK labeling (red color) andIRDye™800-labeled donkey anti-mouse antibodies were added to recognizephospho-ERK labeling (green color). Alexa Fluor® 680-labeledgoat-anti-rabbit antibodies were obtained from Molecular Probes (Eugene,Oreg.). IRDye™ 800CW-labeled donkey-anti-mouse antibodies were purchasedfrom Rockland (Gilbertsville, Pa.). The plates were washed 4 times with0.2% Tween and 0.01% SDS/PBS and scanned using the Odyssey® infraredscanner. Well intensities were quantitated and phospho-ERK signals werenormalized to total ERK signals by the Odyssey® software. Data analysiswas performed using GraphPad Prism (GraphPad Software, San Diego,Calif.).

PAMs can be identified by measuring changes in the phosphorylation ofERK (extracellular receptor kinase) by in-cell western analysis.Compounds with allosteric modulator activity evokeconcentration-dependent increases in ERK phosphorylation. To obtaindata, an α7 NNR agonist such as PNU-282987 (see for example, Hajos etal. J Pharmacol. Exp Ther. 2005; 312: 1213-22) was used as the α7selective agonist. Typical EC₅₀ values in this assay range from about 10nM to about 30 μM. Other α7 nicotinic receptor agonists including2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole,5-[6-(5-methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-1H-indole(published in US 20050065178), various quinuclidine derivatives (see forexample, US 2005/0137204 and US 2005/0245531) and4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-1,4-diaza-bicyclo[3.2.2]nonane andrelated analogs (see for example, WO 2004/029053) also are suitable forthe assay.

(iv) Formalin-Induced Persistent Nociceptive Behavior Assay

Nociception was assessed using the formalin test. The mice were placedin open plexiglass observation chambers for 30 minutes to allow them toacclimate to their surroundings; then they were removed for formalinadministration. Mice were gently restrained while the dorsum of the hindpaw was injected with 20 μL of 2.5% formalin into the plantar surface ofthe right hind paw with a 30-gauge needle. The animals were returned tothe chambers and nociceptive behavior was observed immediately afterformalin injection. Mirrors were placed in each chamber to enableunhindered observation. Nociceptive behavior was quantified as the timelicking the injected paw for continuous 5 minutes (phase 1) and 20-45minutes (phase 2), following formalin injection. Formalin-inducedflinching/licking behavior was biphasic. The initial acute phase (phase1, 0-5 minutes) was followed by a relatively short quiescent period,which was then followed by a prolonged tonic response (phase 2, 20-45minutes).

A reference PAM compound(N-[4-(methylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide (Example20) at 10 mg/kg in 10% DMSO/HBC) administered subcutaneously 10 minutesprior to formalin injection reduced nociceptive behaviors of injectedpaw flicking duration in phase 2 by 68%, indicative of pain relief inthis time period as shown in FIG. 3 indicating that the PAM compoundmight have inhibitory effect on formalin-induced central neuronalsensitization.

(v) Sensory Gating Deficit in DBA/2 mice (N40 Gating) Assay

Sensory gating is a basic central nervous system function thatfacilitates attending to important stimuli, and ignoring irrelevantbackground noise. A practical example of sensory gating is the abilityto follow a conversation at a loud party. An experimental measure ofsensory gating is the suppression of brainwave (evoked potential)responding to repetitive auditory stimuli. Schizophrenic patients cannot suppress responding to repetitive stimuli, and are thereforeconsidered to have sensory gating deficiencies. The DBA/2 mouse is astrain that shows a similar inability to suppress N40 evoked potentialresponding to repetitive auditory stimuli.

DBA/2 mice (20-25 g, 8-10 week, Harlan) were used for gating experimentsand handled in accordance with approved Association for Assessment andAccreditation of Laboratory Animal Care procedures. Mice were given foodand water ad libitum and maintained on a 12-hour light/dark cycle(lights on at 0600 hours).

The surgical techniques employed were similar to those previouslydescribed (Connolly P M, et al. (2003) Brain Res 992(1):85-95). The micewere stereotaxically implanted with tripolar stainless steel wireelectrodes (Plastics One, Roanoke, Va., USA) for Electorencephalogram(EEG) recordings in the CA3 region of the hippocampus. The mice werefirst anesthetized with a solution of 2.8% ketamine (Fort Dodge AnimalHealth, Overland Park, Kans., USA), 0.28% xylazine (PhoenixPharmaceuticals St. Joseph, Mo., USA), and 0.05% acepromazine (PhoenixPharmaceuticals St. Joseph, Mo., USA) at 140 mg/kg of ketamine. A scalpincision was made along the centerline, and the exposed skull wasdisinfected and dried with 3.0% hydrogen peroxide. Three drill holes(#68 drill bit) were made at mediolateral (ML) 1.0, 1.8, and 2.6 mm fromthe central suture. All three holes were located at anteroposterior(AP)-1.8 mm from the bregma; thus, the holes were in a planeperpendicular to the central suture. The hole at ML 2.6 and AP-1.8 mmwas for the electrode directed at the hippocampus. The holes at ML 1.0,AP-1.8 mm and ML 1.8, AP-1.8 mm were for cortical and referenceelectrodes, respectively. The depth of the hippocampal electrode tip wasdorsoventral (DV) 1.65-1.70 mm below the surface of the cortex. Thedepth of the cortical and reference electrodes was DV 0.5 mm from thesurface of the skull, a distance that resulted in the electrode being incontact with, but not penetrating, the cortical tissue. Two additionalholes were drilled in the contralateral skull for placement of anchoringscrews (#00-90, 1/16″). These screws were then driven into the skull,followed by the lowering of the tripolar electrode into the brain with astereotaxic electrode holder. Before completely inserting theelectrodes, a drop of cyanoacrylic glue was placed on the skullunderneath the electrode pedestal. The electrodes and the pedestal werethen completely lowered and the glue was allowed to dry for severalminutes. The pedestal was permanently affixed to the skull with dentalacrylic. The mice were then allowed to recover for at least 4 daysbefore conducting experiments.

To obtain EEG and evoked potentials (EPs), unanesthetized mice wererecorded in acoustically isolated chambers (Med Associates). EEGbiosignals were recorded from freely moving mice using flexible cabletethers and electrical swivels (Plastics One) and amplified withdifferential AC amplifiers (Grass Instrument Division, Astro-Med, WestWarwick, R.I., USA). The EEG was amplified by a factor of 1,000, andband pass filters were set at 1 and 300 Hz. Auditory EPs were generatedby the presentation of 120 sets of paired white noise bursts (5 msdurations) from a speaker within the recording chamber at a distance ofapproximately 15-20 cm from the mouse. The first auditory stimulus ofthe pair, or the conditioning stimulus, was followed 0.5 seconds laterby an identical auditory stimulus, referred to as the test stimulus. Thelength of time between stimulus pairs was 15 seconds. Interpairintervals are generally reported to be 10-15 seconds, a range thatminimizes a potential influence from the previous stimulus pair (Adler LE, et al. (1986) Biol Psychiatry 21:787-798; Connolly P M, et al. (2003)Brain Res 992(1): 85-95; Stevens K E, et al. (1995) Psychopharmacology119:163-170). The volume of auditory stimuli inside the recordingchamber was 65 dB, which was 5 dB above the constant 60-dB backgroundnoise of the recording chambers. No startle response was evoked by thisrelatively low-level stimulation. Data acquisition software (SciWorks,Datawave Technologies, Berthoud, Colo., USA) digitized the EEG signalsat 1,000 Hz, and was set to acquire 1 second of data starting 100 msbefore, and ending 900 ms after, the initial conditioning stimulus. Thesoftware averaged the 120 paired responses into one composite evokedresponse. The measurement of hippocampal EP amplitudes was entirelycomputer automated using peak-finding analysis software (SciWorks). TheEP response to auditory stimuli was identified as a peak deflection at alatency of 10-25 ms after the stimulus, followed by a peak of oppositepolarity at 25-50 ms after the stimulus. The difference in amplitudebetween these two peaks was defined as the N40 amplitude in microvolts(μV). The mouse N40 amplitude, analogous to the human P50, wasdetermined for both the averaged conditioning (CAMP) and test (TAMP) EPsand a ratio was derived between the two responses by dividing the testamplitude by the conditioning amplitude. This calculation, termed theT:C (test:conditioned) ratio, was the measure by which treatments wereassessed for effects on sensory gating. T:C ratios were determined ineach mouse for all recording sessions. DBA/2 mice with control T:Cratios below 0.60, and therefore lacking a sensory gating deficit, wereexcluded from the data analysis.

Drugs were administered 5 minutes before mice were placed into therecording chambers and the initiation of auditory EP recording.Recording of EPs continued for 30 minutes after the recordings began.For every experiment, each mouse was administered all treatments,including a vehicle control, on separate days, with at least 72 hoursbetween treatments. This within-subjects design allowed each mouse toserve as its own control. The treatment order was randomized for eachanimal in all experiments.

Example 20 was dissolved using a vehicle combination of 5% dimethylsulfoxide, 5% Solutol-HS15 (BASF), and 90% phosphate buffered saline(Sigma Chemical). Percentages are of the final solution volume.

The statistical analysis utilized for the dose response was a repeatedmeasures ANOVA followed by a Newman-Keuls multiple comparison post hoctest to identify significant doses.

The T:C ratio is an index of evoked potential suppression; a high ratioindicating little suppression, a low ratio greater suppression. Thus,reduction of T:C ratios by an agent would be considered a therapeuticimprovement of sensory gating. FIG. 4 shows that a PAM (Example 20) at adose of 0.01 μmol/kg lowers T:C ratios in DBA/2 mice by 23.7±8.9% whencompared to vehicle. Therefore, positive allosteric modulation of α7neuronal nicotinic receptors by a PAM (Example 20) has the ability toimprove sensory gating. This suggests that a PAM (Example 20) couldimprove the ability of schizophrenic patients to focus on relevantsensory stimuli, and not be distracted extraneous noise.

(vi) Sigma Receptor Binding Assay

The compounds of the invention are PAMs of α-7 NNR, but not inhibitorsof sigma receptors or do not show any significant binding to sigmareceptors (for examples of sigma receptors ligands and their use, seefor example, U.S. Pat. No. 6,057,371).

Sigma receptors are binding sites that interact with severalpsychoactive agents. Inhibition of specific binding of known ligands tosigma receptors (σ1 and σ2) was used to determine the selectivity oftest compounds. The assays were performed as described previously in theliterature (non-selective σ: Shirayama, Y., et al. Eur. J. Pharmacol.(1993), 237: 117-126. σ1: Ganaphthy, M. E., et al. J. Pharmacol. Exp.Ther. (1999) 289: 251-260. σ2: Bowen, M. D., et al. Mol. Neuropharmacol.(1993) 3: 117-126). The origin of the non-selective σ receptors was ratcerebral cortex. The origin of the σ1 and σ2 receptors were Jurkat cellsand rat cerebral cortex, respectively.

Affinity for the receptors was determined by competitive displacement ofspecific ligands (8 nM of [³H]1,3-di(2-tolyl)guanidine for non-selectiveσ, 8 nM of [³H](+)pentazocine for σ1 and 5 nM of[³H]1,3-di(2-tolyl)guanidine for σ2) by test compounds. Nonspecificbinding was determined by addition of an excess of an unlabelled binder(10 μM haloperidol).

The results are expressed as a percent inhibition of control specificbinding (100−((measured specific binding/control specific binding)×100))obtained in the presence of test compound (10 μM) as shown in Table 1.In addition, Table 1 includes data for the selected compounds from thevoltage-clamp in Xenopus laevis oocytes assay.

TABLE 1 Sigma Receptor Inhibition and Activity in the Voltage-Clamp inXenopus laevis Oocytes Assay % Voltage-Clamp in Inhibition of Xenopuslaevis Binding at Oocytes Example 10 μM EC₅₀ (μM), Number σ σ1 σ2 (%max) 17 8 −1 0.18 (94%) 22 21 −4 1.2 (115%) 43 84 34 >10 (2%) 19 6 5.4(120%) 20 39 0.097 (99%)

Compounds of the invention are PAMs of α7 NNR that can enhance theeffects of a naturally occurring neurotransmitter, acetylcholine, orexogenously administered agonist. Although not being limited by theory,PAMs generally amplify agonist (acetylcholine) responses by (i)attenuating receptor desensitization so that the receptor remains openfor longer duration and/or (ii) by directly amplifying the efficacy ofACh by enhancing maximal receptor activation. In either case, suchcompounds typically boost endogenous transmission of acetylcholine, andcan do so in a temporally and spatially restricted manner since theseeffects will be localized to regions where the α7 receptors areexpressed. Allosteric modulator compounds can modulate the function ofα7 NNRs by enhancing ion channel function as measured by calciumresponses described herein, or other approaches such as current ormembrane potential studies.

In an embodiment, the compounds behave as PAMs in these assays between aconcentration range of about 0.1 nM to about 10 μM. Allostericmodulation of the α7 NNR can trigger key signaling processes that areimportant to effects on memory, cytoprotection, gene transcription anddisease modification. Therefore, the administration of a therapeuticallyeffective amount of a compound of formula (I) to a mammal provides amethod of selectively modulating the effects of α7 NNRs.

It is understood that the foregoing detailed description and examplesare merely illustrative and are not to be taken as limitations upon thescope of the invention. Various changes and modifications to thedisclosed embodiments will be apparent to those skilled in the art. Suchchanges and modifications, including without limitation those relatingto the chemical structures, substituents, derivatives, intermediates,syntheses, formulations and/or methods of use of the invention, may bemade without departing from the spirit and scope thereof. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are hereby incorporated herein by referencein their entireties.

1. A method of treating a disorder or condition selected from the groupconsisting of acute pain, post-surgical pain, inflammatory pain, andneuropathic pain, comprising administering a therapeutically effectiveamount of a compound of formula (I):

wherein, A is —C(O)NH—; R^(a), R^(b), and are independently hydrogen,alkenyl, alkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylsulfonyl,alkylthio, cyano, haloalkoxy, haloalkyl, or halogen; R^(x) at eachoccurrence is independently acyloxy, alkoxy, alkyl, haloalkyl, halogen,or hydroxy; n is 0, 1, 2, 3, or 4; R¹ is hydrogen or halogen; R² and R³are independently hydrogen, alkenyl, alkoxy, alkoxycarbonyl, alkyl,alkylcarbonyl, alkylsulfonyl, alkylthio, cyano, haloalkoxy, haloalkyl,halogen or NR⁵R⁶, provided that at least one of R² or R³ is NR⁵R⁶; R⁵and R⁶ are independently hydrogen, alkenyl, alkoxyalkyl, alkoxycarbonyl,alkyl, alkylcarbonyl, alkylsulfonyl, arylalkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heteroarylalkyl, heterocycle, orheterocyclealkyl; or a pharmaceutically acceptable salt, ester, or amidethereof.
 2. The method of claim 1, further comprising administering incombination with atypical antipsychotics, cholinesterase inhibitors,histamine M3 antagonists, 5HT2_(c) antagonists, 5HT-6 antagonists,dopamine D1 agonists, muscarinic receptor antagonists, potassium channelblockers, α7 NNR ligands, or α4β2 NNR ligands.
 3. The method of claim 1,wherein R¹ is hydrogen or fluorine.
 4. The method of claim 1, wherein R⁵and R⁶ are independently selected from hydrogen, alkyl, alkoxycarbonyl,alkylcarbonyl, alkylsulfonyl, arylalkyl, cycloalkyl, cycloalkylalkyl,haloalkyl, and heteroarylalkyl.
 5. The method of claim 1, wherein thecompound is selected from the group consisting of:N-[4-(acetylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;N-[4-(diethylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;N-{3-[(methylsulfonyl)amino]benzyl}-4-[(trifluoromethyl)thio]benzamide;N-[3-(methylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;N-{4-[(methylsulfonyl)amino]benzyl}-4-[(trifluoromethyl)thio]benzamide;N-[3-(dimethylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;N-{3-[acetyl(methyl)amino]benzyl}-4-[(trifluoromethyl)thio]benzamide;N-[4-(dimethylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;N-(4-aminobenzyl)-4-[(trifluoromethyl)thio]benzamide;N-[4-(methylamino)benzyl]-4-[(trifluoromethyl)thio]benzamide;4-[(difluoromethyhthio]-N-[4-(dimethylamino)benzyl]benzamide; and4-[(difluoromethyhthio]-N-[4-(methylamino)benzyl]benzamide; or apharmaceutically acceptable salt thereof.